Methods and/or Use of Oligonucleotide Conjugates Having Varied Degrees of Labeling for Assays and Detections

ABSTRACT

The present disclosure is directed to methods and/or uses of oligonucleotide conjugates having varied degrees of labeling for assays and detections and related systems and/or kits. Certain methods are directed to a method for detecting one or more biological targets of a sample in a detection assay, comprising: providing a molecular probe, comprising a binding moiety and an oligonucleotide sequence, to a sample comprising one or more biological targets; binding the one or more biological targets with the binding moiety; providing a detectable component to the sample, wherein the detectable component comprises a signal generating moiety conjugated to an oligonucleotide sequence complementary to the oligonucleotide sequence of the molecular probe; hydridizing the oligonucleotide sequence of the target-bound molecular probe to the detectable component; and detecting a signal generated from the hydridized detectable component. Various other embodiments, applications etc. are disclosed herein.

CROSS-REFERENCE

Each of the following documents are incorporated herein by reference inits entirety: U.S. Pat. Nos. 7,462,689; 6,800,728; 7,173,125; 6,686,461;7,102,024; 6,911,535; 6,217,845; 5,753,520; 5,420,285; 5,679,778; and5,206,370. U.S. patent application Ser. No. 11,787,932, filed on Apr.18, 2007, now U.S. Patent Publication No. 2008/0221343, published Sep.11, 2008. U.S. Patent Application No. 61/282,434, filed on Feb. 12,2010. International Application No. PCT/US2001/09252, filed on Mar. 22,2001, now World Publication No. WO 2001/70685; International ApplicationNo. PCT/US2001/023775, filed on Jul. 27, 2001, now World Publication No.WO 2002/010432; International Application No. PCT/US2002/001161, filedon Jan. 16, 2002, now World Publication No. WO 2002/057422. SoluLinkmanual, entitled “Antibody-Oligonucleotide All-in-One Conjugation KitUser Manual”, Catalog No. A-9201-001, January 2010. In addition, each ofthe following provisional applications is incorporated herein byreference in its entirety: U.S. Patent Application Nos. 61/344,931,filed Nov. 22, 2010, and 61/483,186, filed May 6, 2011.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant number5R43AI091340-02 awarded by National Institutes of Health. The governmenthas certain rights in the invention.

FIELD

The present disclosure relates to and may be applied to the methodsand/or uses of oligonucleotide conjugates having varied degrees oflabeling for assays and detections and related systems and/or kits.

BACKGROUND

Current diagnostic tools fail to satisfy certain desired requirementsfor diagnostic assays. For example, current diagnostic tools do notreadily diagnose diseases at earlier stages, yield the informationrequired to direct clinicians to treat patients safely with advancedtherapeutics, quantify the effectiveness of the newmulti-pathogen/component vaccines, correlate information from genesequencing with the protein expression in cells, aid drug developers tobetter understand the activities and toxicities of drugs in developmentfrom pre-clinical to Phase III, allow scientists to study and understandintra- and inter-cellular interactions, and a wide range of otherresearch-based biological and clinical assays.

One of the bottlenecks of current tools is their limit in the number ofassays that can be performed simultaneously or substantiallysimultaneously. For example, in most cases, current protein diagnosticassays only detect 1-10 protein biomarkers simultaneously, orsubstantially simultaneously. In the clinic, for example, the prostatecancer PSA assay measures only a single protein, the prostate-specificantigen protein, and the breast cancer Hercept Test measures only asingle receptor, the Her2 receptor. However, a multitude of interactionsand pathways occur continuously in the cell and many of theseinteractions and pathways are altered in diseased cells. Therefore, inorder to more fully understand the functioning of a cell, including themulti-variant processes conducted within and between normalhealthy-cells, as well as the alterations of these cellular processes invarious disease states, new technologies are needed to track andcorrelate a greater numbers of genetic, protein, and other cellularcomponent changes. Access to this greater amount of information willallow the development of higher content assays, thereby resulting inmore informed clinical decisions and improved patient outcomes.

Bioconjugates have been employed in a wide variety of molecular biologyapplications. For example, bioconjugates are used in biochemical assaysand diagnostic assays to improve assay sensitivity. Bioconjugates, suchas oligonucleotides conjugated to antibodies or enzymes, have been usedas hybridization probes in immunoassays or as probes in the developmentof sensitive nucleic acid-based diagnostic assays. Such conjugates maybe prepared by a variety of methods, such as glutaraldehydecrosslinking, maleimide-thiol coupling, isothiocyanate-amine coupling,hydrazone coupling, oxime coupling, and Schiff base formation/reduction.

Despite the promise that bioconjugates hold in the area of biomedicalresearch, such as improving assay sensitivity, simplifying nucleic aciddetection schemes, clinical studies, development of both in vitro and invivo diagnostic assays as well as in vivo therapies, and the like,bioconjugates have not yet achieved their desired potential in thesemolecular biology, biomedical and diagnostic applications. Thisdeficiency is due, in part, to the inefficient and less thanquantitative preparation of bioconjugates, which may involve multiplesteps and may require, for example, the protein, the oligonucleotide, orboth, to be modified with the appropriate linking moiety and thenpurified before being combined and reacted with each other. Often themodification reaction may have a lengthy reaction time and may result informing an unstable protein or oligomer intermediate that must bepurified and used immediately. For these and other reasons, the yieldsto prepare these bioconjugates are highly variable, and are greatlydependent on what techniques are used. In addition, another issue isthat conventional conjugation chemistries lack the flexiability to costeffectively supply the large number of various conjugates users need.

Another reason that has hindered the widespread use of bioconjugates isthe methods used to purify and isolate bioconjugates. Because of theinefficiencies in the conjugation chemistries used to preparebioconjugates, often the resulting bioconjugate product may requireseveral purification steps to obtain a purified bioconjugate, which canhave a detrimental effect on the stability or activity of the finalbioconjugate, its yield as well as be time consuming and expensive toprepare and/or purify.

Up to this point, the purification of bioconjugates has beenaccomplished using, for example, size exclusion chromatography, oroccasionally, ion exchange chromatography. The requirement forchromatography for purification of bioconjugates has been a significantbarrier for the routine use of bioconjugates, such asantibody-oligonucleotide bioconjugates in diagnostic assays. For theseand other reasons, the costs of preparing and purifying bioconjugateshave been expensive and have been difficult to make with reproducibleresults.

Developments in conjugation chemistry have improved the efficiency ofpreparing bioconjugates. For example, SoluLink™ has developedconjugation chemistry that can be used to prepare abiomolecule-oligonucleotide conjugate, such as antibody-oligonucleotidebioconjugate, with at least 80% efficiency. Accordingly, the preparationof bioconjugates using efficient conjugation chemistries has allowed forthe ability to explore efficient, mild, robust, simple, high yieldingpurification or combinations thereof methods to provide bioconjugates,for example, biomolecule-oligonucleotide conjugates, such asantibody-oligonucleotide bioconjugates, in high yield having high purityto facilitate their use in molecular biology, biomedical, and diagnosticresearch and application.

There still remains a need for methods, systems and or kits that providea more efficient, robust, mild, simple, high-yielding purification orcombinations thereof of such bioconjugates to provide high puritybioconjugates for use in biomedical research and diagnostic assays.There is also a need for methods, systems and/or kits that increase thenumber of assays that can be performed simultaneously or substantiallysimultaneously. The present disclosure is directed to address one ormore of these problems as well as other problems not addressed in thisbackground.

SUMMARY

Certain embodiments provide for methods of detecting one or moremolecular targets in a sample using biomolecule-oligonucleotideconjugates, comprising: i) forming biomolecule-oligonucleotideconjugates at greater than 80% efficiency from at least one or moremodified biomolecules and at least one or more modifiedoligonucleotides, wherein the formed biomolecule-oligonucleotideconjugates comprise one or more detectable components; ii) combining theformed biomolecule-oligonucleotide conjugates with the sample comprisingthe one or more molecular targets; iii) contacting the one or moremolecular targets in the sample with the formedbiomolecule-oligonucleotide conjugates; and iv) detecting the contactedone or more molecular targets.

Certain embodiments provide methods for isolatingbiomolecule-oligonucleotide conjugates, for example,antibody-oligonucleotide conjugates, protein-oligonucleotide conjugates,or peptide-oligonucleotide conjugates, comprising: i) introducing amodified biomolecule into a buffered solution; ii) conjugating themodified biomolecules with at least one modified oligonucleotide atgreater than 80% efficiency to form biomolecule-oligonucleotideconjugates and iii) isolating the biomolecule-oligonucleotide conjugatesfrom the conjugation solution by binding the conjugates to animmobilized binder. As an alternative to using an immobilized binderother isolation techniques may also be used, for example,chromatography, affinity chromatography, size exclusion chromatography,HPLC, reverse-phase chromatography, electrophoresis, capillaryelectrophoresis, polyacrylamide gel electrophoresis, agarose gelelectrophoresis, free flow electrophoresis, differential centrifugation,thin layer chromatography, immunoprecipitation, hybridization, solventextraction, dialysis, filtration, diafiltration, tangential flowfiltration, ion exchange chromatography, hydrophobic interactionchromatography, or combinations thereof.

In certain embodiments, detecting a contacted one or more moleculartargets, for example, a biomolecule-oligonucleotide conjugate contactedone or more molecular targets, may comprise using one or more of thefollowing, comprising: flow cytometry; immunomagnetic cellulardepletion; immunomagnetic cell capture; multiplex bead arrays;microarrays, including antibody arrays, bead arrays, and cellulararrays; solution phase capture; chemiluminescence detection; infrareddetection; microspcopy, imaging; high content screening (HCS);immunohistochemistry (IHC); immunocytochemistry (ICC); in situhybridization (ISH); enzyme immuno-assays (EIA); enzyme linkedimmuno-assays (ELISA); ELISpot; blotting methods, such as a Westernblot, Southern blot, and/or Southwestern blot; labeling insideelectrophoresis systems, labeling on surfaces, and/or labeling onarrays; PCR amplification; elongation followed by PCR amplification;immunoprecipitation, such as co-immunoprecipitation or chromatinimmunoprecipitation; pretargeting imaging or therapeutic agents and/orcombinations thereof. In certain embodiments, a kit and/or system fordetecting one or more molecular targets in a sample, may comprise one ormore prepared, purified and/or isolated molecular probes, such as one ormore biomolecule-oligonucleotide conjugates, for example,antibody-oligonucleotide conjugates, protein-oligonucleotide conjugates,or peptide-oligonucleotide conjugates, one or more prepared, purifiedand/or isolated universal adapters, and/or one or more prepared,purified and/or isolated detectable components, wherein each of themolecular probes, universal adapters, and/or detectable components maycomprise one or more spacer groups. In certain embodiments, the kitand/or system for detecting one or more molecular targets in a samplemay be used in a method of detecting one or more molecular targets in asample.

In certain aspects, the immobilized binder may comprise a metal ionwherein the metal ion is a divalent metal ion, a transition metal ion, adivalent transition metal ion, or combinations thereof. In certainaspects, the transition metal ion is selected from the group comprising:nickel ion, zinc ion, copper ion, iron ion and cobalt ion. In certainaspects, the modified antibody may include a histidine-rich region.

In certain aspects, the immobilized binder may further comprise anorganic chelator selected from the group comprising: iminodiacetic acid,nitrilotriacetic acid and bicinchoninic acid. In certain aspects, theimmobilized binder may comprise an immobilized antibody.

In certain aspects, the modified biomolecule, for example, a modifiedantibody, modified protein, or modified peptide, may comprise amolecular tag incorporated using protein engineering techniques. Incertain aspects, the molecular tag may be selected from the groupcomprising: poly-histidine tag; Flag Tag; Myc-Tag; S-tag; a peptide tag;and/or combinations or derivatives thereof. In certain aspects, theimmobilized antibody may be complementary to the molecular tag that isbound to the modified biomolecule. In certain aspects, the immobilizedantibody may be raised against the molecular tag that is bound to themodified biomolecule. The molecular tag may be a peptide tag. In certainaspects, the immobilized binder may be an antibody raised against theconjugative linker joining the modified biomolecule to the at least onemodified oligonucleotide.

In certain embodiments, the conjugating efficiency of forming amolecular probe, such as a biomolecule-oligonucleotide conjugate, isgreater than about 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 98.5%, or99%. In certain embodiments, the conjugating efficiency of forming amolecular probe, such as a biomolecule-oligonucleotide conjugate, is atleast about 85%, 90%, 92%, 95%, 96%, 97%, 98%, 98.5%, or 99%.

In certain embodiments, the conjugating efficiency of forming adetectable component, comprising one or more signal generating moietiesconjugated directly to an oligonucleotide sequence complementary to theoligonucleotide sequence of a molecular probe or an oligonucleotidesequence complementary to the oligonucleotide sequence of a universaladapter, is greater than about 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%,98.5%, 99%. In certain embodiments, the conjugating efficiency offorming a detectable component, comprising one or more signal generatingmoieties and an oligonucleotide sequence complementary to theoligonucleotide sequence of a molecular probe or an oligonucleotidesequence complementary to the oligonucleotide sequence of a universaladapter, is at least about 85%, 90%, 92%, 95%, 96%, 97%, 98%, 98.5%, or99%. In certain embodiments, the conjugating efficiency of forming adetectable component, comprising one or more signal generating moietiesconjugated indirectly, via a scaffold, to an oligonucleotide sequencecomplementary to the oligonucleotide sequence of a molecular probe or anoligonucleotide sequence complementary to the oligonucleotide sequenceof a universal adapter, is greater than about 80%, 85%, 90%, 92%, 95%,96%, 97%, 98%, 98.5%, or 99%. In certain embodiments, the conjugatingefficiency of forming a detectable component, comprising a scaffold,comprising one or more signal generating moieties, conjugated directlyto an oligonucleotide sequence complementary to the oligonucleotidesequence of a molecular probe or an oligonucleotide sequencecomplementary to the oligonucleotide sequence of a universal adapter, isgreater than about 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 98.5%, or99%.

In certain embodiments, the biomolecule-oligonucleotide conjugates, suchas antibody-oligonucleotide conjugates or protein-oligonucleotideconjugates, comprises on average at least 0.5 modified oligonucleotidesper biomolecule. For example, the modified antibody (e.g.biomolecule-oligonucleotide conjugate) is prepared from an IgG, IgA,IgE, or IgM type antibody. In certain aspects, the modified antibodycomprises an antibody that has been prepared by attaching at least onemoiety comprising a reactive linker capable of conjugating to a modifiedoligonucleotide. This at least one moiety may be attached by a covalentbond. Furthermore, the at least one moiety may comprise a spacer group,for example, a polymerized ethylene oxide, such as PEG or PEO.

In certain aspects, the modified biomolecules, for example, the modifiedantibodies, modified proteins, or modified peptides may be prepared byattaching at least one moiety comprising a reactive linker capable ofconjugating to a modified oligonucleotide. This at least one moiety maybe attached by a covalent bond. The modified biomolecules may furthercomprise a molecular tag. Furthermore, the at least one moiety maycomprise a spacer group, for example, a polymerized ethylene oxide, suchas PEG or PEO.

In certain embodiments, the at least one moiety comprising a reactivelinker may be HyNic (6-HydrazinoNicotinamide). In certain aspects, themodified biomolecule, for example, modified antibody, modified protein,or modified peptide may comprise a HyNic-modified biomolecule (i.e.,covalently modified to display a hydrazinonicotinate reactive moiety).The modified oligonucleotide may also comprise a 4-FB-modifiedoligonucleotide (i.e., covalently modified to display a4-formylbenzamide moiety). In certain aspects, the modified biomoleculemay be a biomolecule that has been modified by attaching at least onemoiety that is a reactive linker capable of conjugating to a modifiedoligonucleotide. The modified biomolecule may further comprise amolecular tag. In certain aspects, the modified biomolecule may comprisean antibody that has been further modified by attaching a biotin thatmay bind to an avidin or a hapten or peptide that may bind to anantibody or a histidine fusion peptide capable of chelating a metal ion.

In certain embodiments, the conjugate may be formed with a covalentlinkage. The covalent linkage may be selected from the group comprising:an amide, an oxime, a hydrazone, a sulfide, an ether, an enol ether, athiol ether, an ester, a triazole and/or a disulfide. The covalentlinkage may comprise a hydrazone. The hydrazone may be abis-arylhydrazone. Furthermore, the covalent linkage may beUV-traceable.

In certain embodiments, the methods of preparing conjugates and themethods of detecting molecular targets disclosed herein may be mild,robust, more efficient, cost effective, simple, and/or combinationsthereof as compared to conventional methods. In addition, such methodsmay provide high purity bioconjugates for use in biomedical applicationsand/or diagnostic assays.

In certain embodiments, the biomolecule-oligonucleotide conjugates, forexample, antibody-oligonucleotide conjugates, protein-oligonucleotideconjugates, or peptide-oligonucleotide conjugates may comprise at leastone modified oligonucleotide. In certain embodiments, thebiomolecule-oligonucleotide conjugates may comprise a composition ofbiomolecule-oligonucleotide conjugates having on average between 1.0 and5, or between 1 and 2.5 modified oligonucleotides conjugated to thebiomolecule. In certain embodiments, the methods disclosed yield atleast between about 30-80%, 40-80%, 40-70%, 60-80% or 70-80% of anisolated biomolecule-oligonucleotide conjugates, with respect tostarting modified biomolecule.

In certain embodiments, the biomolecule-oligonucleotide conjugates, forexample, antibody-oligonucleotide conjugates, protein-oligonucleotideconjugates, or peptide-oligonucleotide conjugates may comprise at leastone or more detectable fluorophores. For example, at least one or atleast two detectable fluorophores. The biomolecule-oligonucleotideconjugates may also comprise at least one or more detectablepoly-fluorophores.

In certain embodiments, the least a portion of thebiomolecule-oligonucleotide conjugates may comprise at least one or moredifferent modified oligonucleotides, such as two different modifiedoligonucleotides.

Certain embodiments provide methods for isolatingbiomolecule-oligonucleotide conjugates comprising: i) conjugating amodified biomolecule with at least one modified oligonucleotide to formbiomolecule-oligonucleotide conjugates, wherein greater than 80% of themodified biomolecules are conjugated; ii) adding the conjugationreaction mixture to a column having a stationary phase comprising abinder that has been immobilized to the stationary phase; iii) bindingthe biomolecule-oligonucleotide conjugates selectively to theimmobilized binder; iv) eluting reaction components away from the boundbiomolecule-oligonucleotide conjugates and v) isolating thebiomolecule-oligonucleotide conjugates by releasing the bound,biomolecule-oligonucleotide conjugates with a displacing agent selectivefor the binder. As an alternative to using an immobilized binder otherisolation techniques may also be used, for example, chromatography,affinity chromatography, size exclusion chromatography, HPLC,reverse-phase chromatography, electrophoresis, capillaryelectrophoresis, polyacrylamide gel electrophoresis, agarose gelelectrophoresis, free flow electrophoresis, differential centrifugation,thin layer chromatography, immunoprecipitation, hybridization, solventextraction, dialysis, filtration, diafiltration, tangential flowfiltration, ion exchange chromatography, hydrophobic interactionchromatography, or combinations thereof.

The preparation methods may be used as part of a kit and/or system ofpreparing, purifying and/or isolating biomolecule-oligonucleotideconjugates, for example, antibody-oligonucleotide conjugates,protein-oligonucleotide conjugates, or peptide-oligonucleotideconjugates. In certain embodiments, the conjugating efficiency isgreater than about 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 98.5%, or99%. In certain embodiments, the methods disclosed yield at leastbetween about 30-80%, 40-80%, 40-70%, 60-80% or 70-80% of an isolatedbiomolecule-oligonucleotide conjugates, with respect to startingmodified biomolecule.

In certain embodiments, the isolation methods may be used as part of akit and/or system of preparing, purifying and/or isolatingbiomolecule-oligonucleotide conjugates, for example,antibody-oligonucleotide conjugates, protein-oligonucleotide conjugates,or peptide-oligonucleotide conjugates. In certain embodiments, theconjugating efficiency is greater than about 80%, 85%, 90%, 92%, 95%,96%, 97%, 98%, 98.5%, or 99%. In certain embodiments, the methodsdisclosed yields of at least between about 30-80%, 40-80%, 40-70%,60-80% or at least between about 70-80% of an isolatedbiomolecule-oligonucleotide conjugates, with respect to startingmodified biomolecule.

In certain embodiments, the detection methods may be used as part of akit and/or system of preparing, purifying and/or isolatingbiomolecule-oligonucleotide conjugates, for example,antibody-oligonucleotide conjugates, protein-oligonucleotide conjugates,or peptide-oligonucleotide conjugates, followed by further utilizing theprepared, purified and/or isolated, biomolecule-oligonucleotideconjugates in an assay, for example, in a detection assay, such as in asingleplex or multiplex assay, for example, a singleplex or multipleximmunodetection assay, for detecting one or more biological targets in asample. In certain embodiments, the conjugating efficiency is greaterthan about 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 98.5%, or 99%. Incertain embodiments, the methods disclosed yield at least between about30-80%, 40-80%, 40-70%, 60-80% or at least between about 70-80% of anisolated biomolecule-oligonucleotide conjugates, with respect tostarting modified biomolecule.

In certain embodiments, the modified biomolecule, for example, amodified antibody, modified protein, or modified peptide, may include ahistidine-rich region.

In certain embodiments, the stationary phase used may comprise a waterinsoluble support. For example, the stationary phase may be agarose,other inert natural, synthetic polymeric materials and/or magnetic.

In certain aspects, the immobilized binder may comprise an immobilizedantibody. In certain aspects, the modified biomolecule may furthercomprise a molecular tag. Furthermore, the immobilized antibody may beselective for the molecular tag that is bound to the modifiedbiomolecule.

In certain embodiments, modified biomolecules are provided. Thesecompounds are prepared, for example, by reaction of a biomolecule ofinterest with one of the functionalities of a bifunctional reagent. Themodified biomolecules are available for conjugation or immobilizationusing the remaining functional group. Biomolecules for use hereininclude, but are not limited to, proteins including antibodies,glycoproteins, peptides, oligonucleotides, RNA and/or DNA.

In certain embodiments, modified solid supports, or substantially solidsupports, are also provided, including, but not limited to, syntheticpolymers, beads, glass, slides, metals and/or particles that have beenmodified by reaction with a bifunctional reagent to afford modifiedsynthetic polymers, beads, latex, glass, slides, metals, includingcolloidal metals and/or particles that possess a hydrazino or oxyaminogroup. Combinations of modified solid supports, or substantially solidsupports, are also contemplated. For example, these modified solid, orsubstantially solid, supports are useful in immobilization ofbiomolecules that possess or are modified to possess a carbonyl group.The immobilized biomolecules may also be used indiagnostic and/ortherapeutic applications.

In certain embodiments, methods for purifying conjugates of biomolecules(for example, biomolecule-oligonucleotide conjugates) may involve metalchelation chromatography that utilizes the interaction of a metal ion,for example, Ni⁺² ion, Zn⁺² ion, Cu⁺² ion, Fe⁺² ion, or Co⁺² ion and theantibody. For example, an aqueous mixture of biomolecule-oligonucleotideconjugates and free, or substantially free, modified-oligonucleotide,may be contacted with a water insoluble stationary phase which has themetal ion chelated to the phase. In certain embodiments, the conjugatechelates with the metal ion whereas neither of the specified freemodified-oligonucleotide chelate. In certain embodiments, subsequentwashing of the phase with a mild buffer may remove, or substantiallyremove, the unbound modified-oligonucleotide. In certain embodiments,the biomolecule-oligonucleotide conjugates may then be eluted from thephase and recovered in a form free, sufficiently free, or substantiallyfree, of unconjugated modified-oligonucleotide.

In certain embodiments, the biomolecule-oligonucleotide conjugates, forexample, antibody-oligonucleotide conjugates, protein-oligonucleotideconjugates, or peptide-oligonucleotide conjugates, may be used indiagnostic and/or therapeutic applications.

Other embodiments, aspects, features, and/or advantages of thistechnology will become apparent from the following detailed descriptionwhen taken in conjunction with the accompanying drawings, which are apart of this disclosure and which illustrate, by way of example, certainprinciples of the disclosed technology.

BRIEF DESCRIPTION OF THE FIGURES

In order to facilitate a more detailed understanding of the nature ofcertain embodiments disclosed herein, exemplary embodiments ofprocesses, systems, kits, preparations, methods, purifications, orcombinations thereof, will now be described in further detail, by way ofexample only, with reference to the accompanying figures in which:

FIG. 1 is a gel electrophoresis loading 400 ng of antibody with SYBRstain, containing the following lanes: Marker (lane 1); SFB-H1A (lane2); HyNic-Bovine IgG (lane 3); Bovine IgG/H1A crude (lane 4) and BovineIgG/H1A purified (lane 5), in accordance with certain embodiments.

FIG. 2 is a gel electrophoresis loading 400 ng of antibody with Lumiteinstain, containing the following lanes: Marker (lane 1); SFB-H1A (lane2); HyNic-Bovine IgG (lane 3); Bovine IgG/H1A crude (lane 4) and BovineIgG/H1A purified (lane 5), in accordance with certain embodiments.

FIG. 3 is a gel electrophoresis loading 500 ng of antibody withCommassie stain, containing the following lanes: Marker (lane 1);SFB-H1A (lane 2); HyNic-Bovine IgG (lane 3); Bovine IgG/H1A crude (lane4) and Bovine IgG/H1A purified (lane 5), in accordance with certainembodiments.

FIG. 4 is a gel electrophoresis with Lumitein stain, containing thefollowing lanes: Marker (lane 1); HyNic-MS anti-FITC 150 ng (lane 2); MSanti-FITC/H1A crude 300 ng (lane 3); MS anti-FITC/H1A purified 300 ng(lane 4) and MS anti-FITC/H1A purified 450 ng (lane 5), in accordancewith certain embodiments.

FIG. 5 is a gel electrophoresis loading 300 ng of antibody with DNASilver stain containing the following lanes: Marker (lane 1); 4FB-H1A(lane 2); Bovine IgG/H1A crude (lane 3); Bovine IgG/H1A purified withDiafiltration spin column 100K (lane 4) and Bovine IgG/H1A purifiedZinc-His-tag-magnetic-bead (lane 5), in accordance with certainembodiments.

FIG. 6 is a gel electrophoresis loading Loading 300 ng of antibody withSilver stain containing the following lanes: Marker (lane 1); 4FB-46mer4FB-oligonucleotide (lane 2); 1:5 MS anti-FITC/46mer 4FB-oligonucleotidecrude (lane 3); 1:5 MS anti-FITC/46mer 4FB-oligonucleotide purified(lane 4); 1:3 MS anti-FITC/46mer 4FB-oligonucleotide crude (lane 5); 1:3MS anti-FITC/46mer 4FB-oligonucleotide purified (lane 6); 1:5 MSanti-FITC/36mer 4FB-oligonucleotide crude (lane 7); 1:5 MSanti-FITC/36mer 4FB-oligonucleotide purified (lane 8); 1:3 MSanti-FITC/36mer 4FB-oligonucleotide crude (lane 9) and 1:3 MSanti-FITC/36mer 4FB-oligonucleotide purified (lane 10), in accordancewith certain embodiments.

FIG. 7 is a gel electrophoresis loading 300 ng of antibody with Silverstain, containing the following lanes: Marker (lane 1); SFB-H1A (lane2); 20× Bovine IgG/DG2A crude (lane 3); 20× Bovine IgG/DG2A purified(lane 4); 30× Bovine IgG/DG2A crude (lane 5); 30× Bovine IgG/DG2Apurified (lane 6); 40× Bovine IgG/DG2A crude (lane 7); 40× BovineIgG/DG2A purified (lane 8); 50× Bovine IgG/DG2A crude (lane 9) and 50×Bovine IgG/DG2A purified (lane 10), in accordance with certainembodiments.

FIG. 8 is a gel electrophoresis of 1.0 μg of antibody with Commassiestain, containing the following lanes: Marker (lane 1); HyNic-MSanti-FITC (lane 2); Purified MS anti-FITC/V3B 19 bp (lane 3); PurifiedMS anti-FITC/H1A 35 bp (Ab 4 mg/ml) (lane 4); Purified MSanti-FITC/Amino-40 40 bp (lane 5); Purified MS anti-FITC/Amino-40 40 bp(lane 6); Purified MS anti-FITC/DG2A 46 bp (lane 7) and Purified MSanti-FITC/Amino-60 60 bp (lane 8), in accordance with certainembodiments.

FIG. 9: Conjugation of HyNic-modified antibody with 4FB-oligonucleotide,in accordance with certain embodiments.

FIG. 10: Magnetic affinity purification of antibody-oligonucleotideconjugate, in accordance with certain embodiments.

FIG. 11: Stage 1: Modification of the oligonucleotide to form a modifiedoligonucleotide, in accordance with certain embodiments.

FIG. 12: Stage 2: Modification of the antibody to form a modifiedantibody, in accordance with certain embodiments.

FIG. 13: Stage 3: Formation of the antibody-oligonucleotide conjugate.Stage 4: Purification of the antibody-oligonucleotide conjugate, inaccordance with certain embodiments.

FIG. 14: is a scheme presenting the HyNic/4FB chemistry used toconjugate oligonucleotides (oligonucleotide barcode tags) to antibodiesand results, in accordance with certain embodiments.

FIG. 15: is a scheme presenting steps used to prepare purifiedbis-arlyhydrazone based antibody-oligonucleotide conjugates and results,in accordance with certain embodiments.

FIG. 16: is a scheme presenting preparation of 1:1antibody-oligonucleotide conjugate via a controlled reduction ofdisulfide bonds in the hinge region of an antibody, followed by reactionof a resultant thiol with a thiol-reactive aromatic hydrazine, MHPH, toform a hydrazine containing adduct, and then conjugated with a4FB-oligonucleotide in the presence of aniline catalyst to form the 1:1antibody-oligonucleotide conjugate, in accordance with certainembodiments.

FIG. 17: is a scheme presenting the preparation of a conjugate betweenan oligonucleotide and a cysteine-containing engineered protein, formedby reacting a cysteine-containing engineered protein with athiol-reactive aromatic hydrazine, MHPH, to form a hydrazine-containingadduct, and then conjugated with a 4FB-oligonucleotide in the presenceof aniline catalyst to form the engineered protein-oligonucleotideconjugate, in accordance with certain embodiments.

FIG. 18: is a general scheme presenting the preparation ofoligonucleotide-signal generators using a HyNic-4FB coupling, inaccordance with certain embodiments.

FIG. 19: is a scheme presenting the preparation of a complementaryoligonucleotide-dextran-polyfluor conjugate with 1:1 oligonucleotide todextran stoichiometry, in accordance with certain embodiments.

FIG. 20: (Left) is a schematic representation of the hybridization of anantibody-oligonucleotide conjugate (A), with a complementaryoligonucleotide-fluorescently labeled scaffold (B), to form thehybridization product (C). (Right) Polyacrylamide gel resultsdemonstrating the hybridization of two oligonucleotide conjugates (lanes3 and 5) as compared to their respective complementaryoligonucleotide-dextran scaffold conjugates (lanes 4 and 6), inaccordance with certain embodiments.

FIG. 21: is a schematic representation of conjugate self assembly,wherein a series of antibody-oligonucleotides (A), and a series ofcomplementary oligonucleotide-signal generators (B), self-assemble viahybridization to form hybridrization products (C), in accordance withcertain embodiments.

FIG. 22: is a schematic representation of a two-plex flow cytometryexperiment mediated by self assembly by hybridization ofantibody-oligonucleotide conjugates bound to their respective antigens(Probe) followed by hybridization to their complementaryoligonucleotide-signal generator conjugates (Detect), in accordance withcertain embodiments.

FIG. 23: is flow cytometry results demonstrating detection of CD4 onliving cells using α-CD4 antibody-HyLk1 conjugate+HyLk1′-R-phycoerythrinconjugate, in accordance with certain embodiments.

FIG. 24: is flow cytometry results demonstrating detection of CD4 onliving cells using α-CD4 antibody-HyLk1 conjugate+HyLk1′-allophycocyaninconjugate, in accordance with certain embodiments.

FIG. 25: is flow cytometry results demonstrating detection of CD4 onliving cells using α-CD4 antibody-HyLk1 conjugate+HyLk1′-poly-Dy490conjugate, in accordance with certain embodiments.

FIG. 26: is comparing flow cytometry detection results of either anα-CD4 antibody (A) or an α-CD8 antibody (B) on living cells, wherein theα-CD4 or α-CD8 antibodies are directly labeled with FITC or are labeledvia antibody-HyLk1 conjugate and HyLk1′-poly-Dy490 conjugates, inaccordance with certain embodiments.

FIG. 27: in both (A) and (B) are results demonstrating absense ofcrosstalk between antibody-oligonucleotide conjugates andnon-complementary oligonucleotide fluorophore conjugates, in accordancewith certain embodiments.

FIG. 28: is results demonstrating a time course experiment (A)-(E) oflabeling by hybridization between an antibody-oligonucleotide conjugateand complementary oligonucleotide-polyfluor conjugate, with graph (F)showing the results of (A)-(E) superimposed, in accordance with certainembodiments.

FIG. 29: is results of an experiment (A)-(D) titrating the amount ofcomplementary oligonucleotide-R-phycoerythrin conjugate sufficient toproduce a desired signal, in accordance with certain embodiments.

FIG. 30: (Top; A-B) is flow cytometry results demonstrating detection ofCD19 on living cells using α-CD19 antibody-HyLk3conjugate+HyLk3′−poly-DY591 and (Bottom; C-D) results demonstratingdetection of CD8 on living cells using α-CD8 antibody-HyLk2conjugate+HyLk2′-poly-DY549, in accordance with certain embodiments.

FIG. 31: is a 5-Plex flow cytometry experiment (A)-(D) using 5commercially available antibody-fluorophore conjugates (Directly LabeledAntibody conjugates). In this experiment, the same panel of antibodieswas used as in FIG. 32 as a reference example to compare the performanceof each panel, in accordance with certain embodiments.

FIG. 32: is a 5-Plex flow cytometry experiment (A)-(D) using 5 differentoligonucleotide-antibody conjugates (Molecular Probes) followed byaddition of the complementary oligonucleotide-polyfluor conjugates(Detectable Components), in accordance with certain embodiments. Thepattern of immune reactivity is to be compared with the panel in FIG. 31for the same antibodies but using direct fluorescent conjugates.

FIG. 33: is a scheme presenting hybridization-mediated immunomagneticseparation of CD4⁺ cells, with (A) a binding of α-CD4-oligonucleotideconjugate and (B) a hybridization to a complementaryoligonucleotide-immobilized paramagnetic bead and attraction by amagnet, in accordance with certain embodiments.

FIG. 34: is a scheme presenting hybridization-mediated immunomagneticseparation of CD4⁺ cells and their release by strand displacement of thehybridization by a displacement oligonucleotide such as a peptidenucleic acid (PNA) or a locked nucleic acid (LNA), in accordance withcertain embodiments.

FIG. 35: is a schematic representation of isolation of an epithelialcell, such as a circulating cancer cell, in a microfluidic device usingα-Epithelial Cell adhesion molecule antibody-oligonucleotide conjugatesand their complementary oligonucleotides immobilized in a microfluidicchannel while allowing lymphocytes to escape, in accordance with certainembodiments.

FIG. 36: (Left) is a schematic representation of detection of an antigenin a Western Blot experiment using an antibody-oligonucleotideconjugate/complementary oligonucleotide-signal generator pair. (Right)Results comparing a classical Western Blot experiment detecting tubulinand a likely non-specific band using a primary α-tubulinantibody/secondary antibody-HRP conjugate to a Western Blot preparedusing an α-tubulin antibody-oligonucleotide conjugate and complementaryoligonucleotide-HRP conjugate, in accordance with certain embodiments.

FIG. 37: is a schematic representation of an Universal Adapter protocol,wherein each antibody is conjugated to oligonucleotides of the sameUniversal sequence, but each is then hybridized to a distinct UniversalAdaptor prior to their use in a multiplexed experiment, to allow them tobe differentiated, and then performing a detection experiment, whereinthe signal generator is linked to the 5′-end of theoligonucleotide-signal generator conjugate, in accordance with certainembodiments.

FIG. 38: is a schematic representation of a Universal Adapter protocol,wherein each antibodies is conjugated to oligonucleotides of the sameUniversal sequence, but each is then hybridized to a distinct UniversalAdaptor prior to their use in a multiplexed experiment, to allow them tobe differentiated, and then performing a detection experiment, whereinthe signal generator is linked to the 3′-end of theoligonucleotide-signal generator conjugate, in accordance with certainembodiments.

FIG. 39: is infrared fluorescent Western Blot detection resultsperformed using an α-tubulin antibody then detected by an IR800-dyelabeled secondary anti-immunoglobulin antibody compared to using anα-tubulin antibody-HyLk1 oligonucleotide conjugate an HyLk2′oligonucleotide-dextran-poly-IR800 dye conjugate mediated byhybridization to an HyLk1′-HyLk2 adaptor oligonucleotide as presented inFIG. 32, in accordance with certain embodiments.

FIG. 40: is flow cytometry results demonstrating the use of an adaptermethod, showing (A) a positive control: Binding of a molecular probe andthen hybridization with its complement detectable component, in theabsence of an adapter; (B) use of an adapter: Binding of a molecularprobe, hybridization with a complementary sequence on an adapter, andthen hybridization of a second sequence on the adapter withcomplementary sequence on a detectable component; and (C) a negativecontrol: Binding of a molecular probe and addition of an adapter havinga non-complementary sequence to the detectable component, showing nosignal when analyzed by flow cytometry; in accordance with certainembodiments.

FIG. 41: is a schematic representing steps in anantibody-oligonucleotide directed multiplex bead array protocol, wherein(A) the antigen is captured by an antibody-oligonucleotide conjugate,(B) complementary oligonucleotide-beads are added and theantigen/antibody-oligonucleotide complex is captured by hybridization,(C) a biotinylated detector antibody conjugate is added followed by (D)addition of Streptavidin-R-PE conjugate signal detector, to form theresulting capture adduct (E), in accordance with certain embodiments.

FIG. 42: is a schematic representation of a multiplexantibody-oligonucleotide/complementary oligonucleotide-bead conjugateself-assembly, in accordance with certain embodiments, such as in FIG.41.

FIG. 43: is a scheme representing steps in an antibody-oligonucleotidedirected bead array protocol, wherein (A) the antigen is captured by twomolecular probes, comprising an antibody-oligonucleotide conjugate fordetection and an antibody-oligonucleotide conjugate for capture to forma “sandwich immune complex,” (B) a complementary oligonucleotide-bead isadded and the sandwich immune complex is captured by hybridization withthe oligonucleotide barcode sequence from the antibody-oligonucleotideconjugate for capture, (C) addition of a detectable component comprisingan oligonucleotide sequence complementary to the antibody-conjugate fordetection, resulting in (D) the hybridized formation of a fully capturedand detectable complex, in accordance with certain embodiments.

FIG. 44: is a scheme representing steps in an antibody-oligonucleotidedirected bead array protocol, to simultaneously, or substantiallysimultaneously, detect and quantify auto-antibodies from a sample anddetect and quantify the isotype response in a serology assay, wherein(A) an antigen conjugate and an anti-isotype specific antibody conjugateare added to a sample wherein the anti-antigen antibody is captured byboth oligonucleotide conjugates and (B) added to mixture arecomplementary detectable components (distinct bead conjugates) resultingin the capture of the complex by hybridization to the captured antigen,(C) the complex is detected by the addition of complementary detectablecomponents, in accordance with certain embodiments.

FIG. 45: is a scheme representing the steps in anantigen-oligonucleotide directed bead array protocol wherein (A) theanti-antigen antibody is captured by its cognate antigen-oligonucleotideconjugate, (B) complementary oligonucleotide-beads are added and theanti-antigen antibody/antigen-oligonucleotide complex (“immune complex”)is captured by hybridization, (C) a biotinylated detector antibodyconjugate is added followed by (D) addition of Streptavidin-R-PEconjugate signal detector, resulting in (E) the formation of a capturedand detectable complex, in accordance with certain embodiments.

FIG. 46: is a schematic representation of a multiplexantigen-oligonucleotide/complementary oligonucleotide-bead conjugateself-assembly, in accordance with certain embodiments, such as in FIGS.43, 44 and 45.

FIG. 47: is a schematic representing the steps in anantigen-oligonucleotide directed bead array protocol wherein (A) theanti-antigen antibody or antibodies, i.e., IgG, IgM, IgA, or IgE, iscaptured by its cognate antigen-oligonucleotide conjugate, (B)complementary oligonucleotide-beads are added and the anti-antigenantibody/antigen-oligonucleotide complex (“immune complex”) is capturedby hybridization, (C) antibody-oligonucleotide conjugates specific forthe anti-antigen antibodies (“anti-antigen antibody detectors”) areadded followed by (D) the addition of complementaryoligonucleotide-signal detector conjugates that recognize theirrespective oligonucleotide sequences on the anti-antigen antibodydetectors allowing identification of the antibody isotype response, inaccordance with certain embodiments.

FIG. 48: is a schematic representation of the use ofantibody-oligonucleotide conjugate/complementary oligo bead conjugatesin an Immunoturbidity assay, wherein (A) a pair ofantibody-oligonucleotide conjugates (“capture antibody-conjugates”)directed to independent epitopes of an antigen are added to a sample,(B) allowed to bind to form an “immune complex,” (C) the mixture isadded to beads conjugated to oligonucleotides complementary to theoligonucleotides on the capture antibody-conjugates, and (4) allowed tohybridize leading to crosslinking by hybridization, in accordance withcertain embodiments.

FIG. 49: is a schematic representing steps in anantibody-oligonucleotide directed ELISA or planar array protocol wherein(A) an oligonucleotide is immobilized (e.g., on a 96-well plate orplanar array surface), (B) the antigen is captured by anantibody-oligonucleotide conjugate, (C) the sample containing theantigen/antibody-oligonucleotide complex is incubated with the surfaceand captured by hybridization, (D) a biotinylated detector antibodyconjugate is added to bind the captured antigen, followed by (E)addition of Streptavidin-HRP conjugate signal detector, in accordancewith certain embodiments.

FIG. 50: is a schematic representing steps in an antigen-oligonucleotidedirected ELISA or planar array protocol wherein (A) an oligonucleotideis conjugated to BSA and the oligonucleotide-BSA conjugate isimmobilized (e.g., on a 96-well plate or planar array surface), (B) theantibody is captured by an antigen-oligonucleotide conjugate, (C) thesample containing the antibody/antigen-oligonucleotide complex isincubated with the surface and captured by hybridization, (D) abiotinylated detector antibody conjugate is added to bind the capturedantibody, followed by (E) addition of Streptavidin-HRP conjugate signaldetector, in accordance with certain embodiments.

FIG. 51: is a schematic representing similar steps as those in FIG. 50,but wherein the steps are directed to an ELISA or planar array protocolin a serology-based assay to detect anti-antigen antibodies, inaccordance with certain embodiments.

FIG. 52: is showing results of an immunocytochemistry experiment toexamine tubulin distribution in cells, wherein an α-tubulin HyLk1oligonucleotide conjugate molecular probe is imaged using an HyLk1′complementary oligonucleotide-poly-Dy490 fluorophore detector and thefluorescein channel of an epifluorescence microscope as in (A) while theHyLk1′-poly-Dy490 probe alone yields no similar image, according tocertain embodiments.

FIG. 53: is showing imaging results of the distribution of tubulin andthe distribution of phosphotyrosine-containing proteins usingantibody-oligonucleotide conjugates and their respective complementarydetectors applied in mixtures, wherein a mixture of an α-tubulin-HyLk1probe and α-phosphotyrosine-HyLk2 probe are applied and then detected bya mixture of HyLk1′-poly-Dy490 and HyLk2′-poly-Dy549, allowing theirdistribution in (A) cells imaged in brightfield to be distinguished asin (B) using a fluorescein filter set and (C) using a rhodamine filterset, according to certain embodiments.

FIG. 54: is a schematic representation of the process of preassemblyusing pairs of complementary oligonucleotides in which a plurality ofmolecular probes and a plurality of detectable components arehybridized, i.e., preassembled, to form a plurality of preassembledmolecular probe-detectable component hybrids, prior to contacting with asample comprising one or more molecular targets. The preassembly processmay be completed by individual preassembly, followed by pooling, thencontacting with the sample, or alternatively, may be completed bypooling the plurality of molecular probes and plurality of detectablecomponents, preassembling by hybridization, and then contacting thepooled preassembled hybrids with the sample.

FIG. 55: Are flow cytometry results demonstrating the process ofpreassembly on mouse splenocytes, as compared to sequential assembly inwhich the probes are applied in a first step and then the detectors in asecond step. The flow cytometry dot plots on the left (labeled“sequential 5-plex”), illustrate the sequence of using the αCD4-HyLk1,αCD8-HyLk2, αCD19-HyLk3, αCD43-HyLk4 and αCD62L-HyLk5antibody-oligonucleotide conjugates as applied to the mouse splenocytes,followed by washing, then hybridizing with the HyLk1′-Dy490,HyLk2′-Dy549, HyLk3′-Dy591, HyLk4′-Dy649 and HyLk5′-Dy405 detectors, andthen analyzed by flow cytometry. The flow cytometry dot plots on theright (labeled “Preassembled 5-plex (in pool)”), illustrate thecombining the single pool of antibody-oligonucleotide conjugatescomprising αCD4-HyLk1, αCD8-HyLk2, αCD19-HyLk3, αCD43-HyLk4 andαCD62L-HyLk5, preassembling via hybridization with the complementarypolyfluor signal generating moiety conjugates HyLk1′-Dy490,HyLk2′-Dy549, HyLk3′-Dy591, HyLk4′-Dy649 and HyLk5′-Dy405, thencontacting the preassembled hybrids with mouse splenocytes as a pooledmixture, followed by binding, washing, and then analyzed by flowcytometry.

FIG. 56: is results comparing the use of two alternative methods ofpreassembly, the left (labeled “Preassemble in pool, then add tocells”), and the right (labeled “Preassemble one-by-one, pool, add tocells”). The comparable results of the two alternative methods ofpreassembly suggest that either these alternatives, or other preassemblyprotocols, may be followed with similar success.

FIG. 57: illustrates several schemes that may be considered to addressand/or decrease the potential for cross-talk, such as in panel A, byhybridizing the oligonucleotide sequence of a non-hybridized molecularprobe with an unconjugated complementary oligonucleotide, or asillustrated in panel B, by hybridizing the complementary oligonucleotidesequence of a non-hybridized detectable component with an unconjugatedoligonucleotide, or as illustrated in panel C, stabilizing the duplexesformed by the preassembly hybridization of the molecular probe(s) andthe detectable component(s) with natural or synthetic minor groovebinding agents or with natural or synthetic intercalating agents.

FIG. 58: illustrates a preassembly process utilizing a universaloligonucleotide conjugated to a panel of molecular probes that areindividually combined with a universal oligonucleotide complementconjugated to a panel of signal generating moieties. The preassembledmolecular probe-signal generating moiety hybrids may then be stabilizedwith unconjugated oligonucleotides or duplex stabilizers, followed bycontacting with a sample comprising one or more molecular targets oranalytes to perform one or more assays.

FIG. 59: illustrates the stabilizing and then pooling of a panel ofindividually preassembled antibody-signal generating moiety hybrids,followed by contacting with a sample comprising one or more moleculartargets or analytes to perform an assay.

FIG. 60: illustrates a preassembly process utilizing a universaloligonucleotide conjugated to a panel of molecular probes, such as apanel of monoclonal antibodies that are individually combined with auniversal oligonucleotide complement conjugated to a panel of barcodedparticles, that may be used in a flow cytometry-based multiplexedimmunodetection assays. The preassembled antibody-bead hybrids may thenbe stabilized with unconjugated oligonucleotides or duplex stabilizers.The individual stabilized preassembled antibody-bead hybrids may then becontacted with a sample comprising one or more molecular targets oranalytes to perform one or more assays.

FIG. 61: illustrates the stabilizing and then pooling of a panel ofindividually preassembled antibody-bead hybrids, followed by contactingwith a sample comprising one or more molecular targets or analytes toperform an assay.

FIG. 62: illustrates the preassembly of barcoded oligonucleotidedetectable components, comprising a first oligonucleotide sequence, suchas a 20-oligonucleotide universal sequence, comprising anoligonucleotide sequence complementary to a universal oligonucleotideconjugated to a molecular probe, and a second oligonucleotide sequence,such as a 20-oligonucleotide unique sequence, comprising a sequence thatis unique to that detectable component.

FIG. 63: illustrates the pooling of the individually preassembledmolecular probe-barcoded oligonucleotide detectable component hybrids,contacting with a sample comprising one or more molecular targets oranalytes, followed by washing, disassociation of the hybrids, elution,and analysis.

FIG. 64: illustrates the process of preassembly with the use of one ormore universal adapters.

FIG. 65: illustrates the use of detectable components that comprise anoligonucleotide sequence that has been chemically modified withfluorescent moieties to enable detection by fluorescence resonanceenergy transfer (FRET).

FIG. 66: illustrates the preassembly of one or more molecular probeswith one or more supports to form a preassembled affinity matrix ormaterial. The preassembled affinity matrix or material may then be used,for example, to affinity capture, purify, and then release one or moremolecular targets, such as one or more biomolecular targets, each as acomplex bound to the particular molecular probe.

FIG. 67: illustrates a molecular probe conjugated to a universaloligonucleotide may be combined with a sample comprising one or moremolecular targets to bind the one or more targets, which may then becaptured by a complementary oligonucleotide conjugated to a support viahybridization.

FIG. 68 illustrates the Flow cytometric results of the evaluation ofcomplementary oligonucleotide-dextran-polyfluor conjugate detectors withincreasing number of fluors/dextran scaffold, according to certainembodiments.

FIG. 69 illustrates an exemplary scheme for the incorporation of4FB-moiety using a 4FB-phosphoamidite (X) on the 5′-end of anoligonucleotide during its solid phase synthesis, according to certainembodiments.

FIG. 70 illustrates an exemplary schematic representation of the processused to purify 4FB-oligonucleotides, according to certain embodiments.

FIG. 71A shows results for conjugation of a 4FB-20mer oligonucleotide (4mol equiv) to a HyNic-modified antibody, according to certainembodiments.

FIG. 71B shows results for a crude a 4FB-20mer oligonucleotide (5 molequiv) to a HyNic-modified antibody, according to certain embodiments.

FIG. 72 shows a PAGE gel of the conjugation of a 20mer4FB-oligonucleotide purified (Lane 2) to a HyNic-Peg2-9mer peptide (1.5mol equiv (Lane 3) and 3.0 mol equiv (Lane 4)), according to certainembodiments.

FIG. 73 shows the flow cytometric results analyzingantibody-oligonucleotide conjugates hybridized to its complementaryoligonucleotide/dextran/poly-Dy490 detector with respect to the numberof oligonucleotides conjugated to the antibody, according to certainembodiments.

FIG. 74 shows Immunoturbidity assay results per certain embodiments.

FIG. 75 shows the flow cytometry results of the capture and detection ofα-BSA antibody using BSA-HyLk1 conjugate immobilized on Compel-HyLk1′beads from 366 to 0.36 ng.

FIG. 76 illustrates an exemplary step wise protocol that may be used tocapture and detect an antigen from a biological sample, according tocertain embodiments.

FIG. 77 illustrates an exemplary schematic representation of selfassembly, according to certain embodiments.

FIG. 78 illustrates an exemplary schematic representation ofpreassembly, according to certain embodiments.

FIG. 79 presents the flow cytometry results of the binding of α-CD8 20,30 and 40mer hybrids to CD8+ splenocytes, according to certainembodiments.

FIG. 80 is an exemplary scheme presenting hybridization-mediatedimmunomagnetic separation of Her2+ cells from a complex mixture of cell,by (A) a binding of a herceptin-oligonucleotide conjugate and (B) ahybridization to a complementary oligonucleotide-immobilizedparamagnetic bead and attraction by a magnet, in accordance with certainembodiments.

FIG. 81 is the flow cytometric results of the isolation of Her2+ cellsfrom a complex mixture of cells using Herceptin directly immobilized onmagnetic beads to a herceptin-oligonucleotide conjugate followed byaddition of magnetic beads immobilized with the complementaryoligonucleotide, according to certain embodiments.

FIG. 82 is an exemplary scheme presenting hybridization-mediatedimmunomagnetic separation of CD4⁺ cells and their release by stranddisplacement of the hybridization by a displacement oligonucleotide suchas a peptide nucleic acid (PNA) or a locked nucleic acid (LNA), inaccordance with certain embodiments.

FIG. 83: is a schematic representation of isolation of an epithelialcell, such as a circulating cancer cell, in a microfluidic device usinga Herceptin antibody-oligonucleotide conjugate and its complementaryoligonucleotide immobilized in a microfluidic channel while allowinglymphocytes to escape, in accordance with certain embodiments.

DETAILED DESCRIPTION

The following description is provided in relation to several embodimentswhich may share common characteristics and features. It is to beunderstood that one or more features of one embodiment may be combinablewith one or more features of the other embodiments. In addition, asingle feature or combination of features in certain embodiments mayconstitute additional embodiments.

In this specification, the word “comprising” is to be understood in its“open” sense, that is, in the sense of “including”, and thus not limitedto its “closed” sense, that is the sense of “consisting only of”. Acorresponding meaning is to be attributed to the corresponding words“comprise”, “comprised” and “comprises” where they appear.

The subject headings used in the detailed description are included onlyfor the ease of reference of the reader and should not be used to limitthe subject matter found throughout the disclosure or the claims. Thesubject headings should not be used in construing the scope of theclaims or the claim limitations.

CERTAIN TERMS AND DEFINITIONS

The term “molecular probe” may refer to a conjugate, for example, abioconjugate, comprising a binding moiety and an oligonucleotide, forexample a binding moiety conjugated to an oligonucleotide. The molecularprobe may comprise a biomolecule conjugated to an oligonucleotide, forexample, a biomolecule-oligonucleotide conjugate, such as anantibody-oligonucleotide conjugate, an (antibodyfragment)-oligonucleotide conjugate, a protein-oligonucleotideconjugate, or a peptide-oligonucleotide conjugate. The molecular probemay comprise a binding moiety, such as a biomolecule, conjugated to oneor more oligonucleotides, for example, conjugated to twooligonucleotides, three oligonucleotides, or four oligonucleotides.

The term “binding moiety” may refer to a moiety, molecule, or substancethat binds at least one target in a sample. For example, a bindingmoiety may comprise a biomolecule, a synthetic molecule, a biopolymer,or a portion of the biomolecule, synthetic molecule, or biopolymer.Suitable binding moiety may include, but is not limited to, an antibody,antibody-fragment, such as a single chain variable fragment (“scFv”),genetically-modified antibody, genetically-modified antibody-fragment,antigen, a protein, a peptide, a carbohydrate, a nuclear receptor, asmall molecule, a drug or drug-like molecule, or combinations orderivatives thereof. The binding moiety may be capable of recognizingand binding a target. The binding moiety may also comprise a specificbinding affinity for a target. The binding moiety may comprise one ormore oligonucleotides, for example, may be conjugated to one or moreoligonucleotides. The binding moiety may comprise a spacer group. Thebinding moiety may also comprise a universal adapter.

The term “biomolecule” may refer to a compound found in nature, aderivative of a compound found in nature, a synthetically modifiedanalog of a compound found in nature, a genetically engineered analog ofa compound found in nature, a genetically engineered modified analog ofa compound found in nature. For example, biomolecules may be and/orinclude, but are not limited to, proteins; antibodies;antibody-fragments; haptens; glycoproteins; cell-membrane proteins;enzymes, such as alkaline phosphatase, β-galactosidase, horseradishperoxidase, or urease; peptides; peptide nucleic acids (PNAs); lockednucleic acids (LNAs); genetically engineered peptides; geneticallyengineered proteins; genetically engineered antibodies; geneticallyengineered antibody-fragments; oligonucleotides; RNA; DNA;saccharide-containing molecules; monosaccharides; disaccharides;trisaccharides; oligosaccharides; polysaccharides, such as dextran;small molecules, including drug-like molecules; drugs; antigens, such astumor antigens; pathogens; toxins; polymers, including biopolymersand/or dendrimers; nuclear receptors; nuclear receptor substrates and/orligands; cytokines; epitopes, including peptide epitopes, antigenepitopes, and/or pathogen epitopes; enzyme substrates; and/orcombinations or derivatives thereof.

The term “biopolymer” may refer to a compound found in nature, aderivative of a compound found in nature, a synthetically modifiedanalog of a compound found in nature, a genetically engineered analog ofa compound found in nature, a genetically engineered modified analog ofa compound found in nature, wherein the biopolymer may be made up ofmonomeric units. For example, biopolymers may be and/or include, but arenot limited to, oligonucleotides, RNA, DNA, peptides, peptide nucleicacids (PNAs), locked nucleic acids (LNAs), derivatized forms of nucleicacids, proteins including antibodies, glycoproteins, enzymes,oligosaccharides, and/or derivatives thereof. Examples of monomericunits include, but are not limited to, nucleotides, nucleosides, aminoacids, PNA monomers, monosaccharides, and derivatives thereof.

The term “molecular tag” may refer to a peptide sequence that isattached to a molecule. For example, the molecular tag may be a peptidesequence that is recognized as an antigen by an antibody. The moleculartag may include, but is not limited to, a poly-histidine tag, forexample, a Flag Tag, a Myc-Tag, an S-tag, a StrepTag, a calmodulin tag,or a peptide tag that an antibody has been raised against. The moleculartag may be attached to a molecule by synthetic means, by utilization ofrecombinant methodologies, genetic engineering, or combinations thereof.The molecular tag may be a cloned short stretch of polyhistidines thatis attached either onto the amino or carboxy terminus of a protein. Themolecular tag may be recognized by an antibody. The molecular tag mayform a chelate with a metal ion. For example, the molecular tag may be apoly-histidine tag or a tetra-cysteine tag that may form a chelate witha metal ion. Alternatively, the molecular tag may be a protein domain orother folded peptide domain or domains. For example, the molecular tagmay be a glutathione-S-transferase tag, a HaloTag®, a maltose bindingprotein-tag, a monomeric avidin domain, a protein Aimmunoglobulin-binding Z domain, a green fluorescent protein-tag, or athioredoxin-tag. The protein domain may bind to another protein, apeptide or a ligand, by non-covalent or by covalent means.

The term “modified” may refer to a modification of a molecule, such as abiomolecule or a biopolymer, either by chemical synthesis,bio-engineering, or the like. The molecule may be modified by theattachment of a moiety, for example by a covalent bond, onto themolecule, such that once attached, the now modified molecule is capableof reacting with another molecule to form a conjugate. The moiety mayattach to the molecule to form the modified molecule includes a reactivegroup, or a linkable group available to link, i.e., conjugate, toanother complementary reactive group attached to another molecule. Themodified molecule may comprise a reactive group that is protected, andrequires deprotection before being available to link, i.e., conjugate,to another reactive group attached to another molecule. The modificationof a molecule may further comprise attaching a spacer group, a moleculartag, a fusion protein comprising a histidine rich region, orcombinations thereof.

The term “bioconjugate” may refer to a conjugate of at least twobiomolecules, of at least two biopolymers, or at least one biomoleculeand at least one biopolymer. The bioconjugate may also include one ormore linkages between the individual components that have beenconjugated. The bioconjugate may also include one or more spacer groupsbetween the one or more linkages joining the one or more individualcomponents, or the spacer group may be between the individual componentand the linkage. For example, the spacer group may include, but is notlimited to an ethyleneoxide moiety, a polymer formed fromrepeating—(—CH₂—CH₂O—)— moieties, PEG, or PEO.

The term “conjugate” may represent a compound containing at least twocomponents linked together. The individual components may be linkeddirectly through one or more covalent bonds, or one or more ionic bonds,or by chelation, or mixtures thereof. The linkage, or conjugation, mayinclude one or more spacer groups between the one or more linkagesjoining the one or more individual components, or may be between theindividual component and the linkage. The individual components that maybe linked together may include, but is not limited to biologicallyderived biopolymers, modified biopolymers, biologically derivedbiomolecules, and synthetically derived molecules. For example, theconjugate may comprise a first component, such as a protein, that may belinked, i.e., conjugated, directly through one or more covalent bonds toa second component, such as an oligonucleotide, to form a conjugate. Theconjugate and/or the linkage of the conjugate may be stable tothermolysis, stable to hydrolysis, may be biocompatible, or combinationsthereof.

The term “hybrid” may refer to a multicomponent composition formed bybringing together at least two conjugates, formed as disclosed hereinand comprised of at least one probe conjugate and at least one detectorconjugate. A probe conjugate, for example, may be comprised of anantibody, binding protein, nucleic acid aptamer, ligand, chemicalcompound and/or other molecule specific to a target. In certainembodiments, a hybrid would typically be comprised of a single probe anda single detector wherein the oligonucleotide component of the probe iscomplementary to the oligonucleotide component of the detector. Certainembodiments are directed to when the probe conjugate is an antibodyconjugated to two or three oligonucleotides. Alternatively, the probeconjugate may be an antibody conjugated to one, one to two, two tothree, two to four, three to five, four to seven, or five or moreoligonucleotides. Alternatively, the probe conjugate may be an antibodyconjugated to one oligonucleotide sequence, two sequences, threesequences or three or more sequences. In certain embodiments, a detectoris comprised of a detectable component, inclusive of a scaffold, anucleic acid, and/or other chemical compounds, to which is appendedfluorescent or other optically active chemical groups, or bindingmoieties such as biotin or digoxigenin, or peptides such as epitopes, orproteins such as avidin or phycoerythrin or enzymes, or particles suchas quantum dots, or colloidal gold, or latex beads, or surfaces such asglass or polymers or plastics. In certain embodiments, the detector is adextran or other scaffold covalently modified with multiple fluorophoresand a single oligonucleotide. In other embodiments, the detector is anenzyme, fluorescent protein, or other protein conjugated to a singleoligonucleotide. Further, a hybrid is formed when complementaryoligonucleotides on a probe conjugate and a detector conjugate areallowed to anneal or hybridize, based on complementary and base pairing,to form a double-stranded nucleic acid linkage between the probecomponent and the detector component. In certain embodiments a smallvolume, around 10, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 80, 90microliters or less, of a solution of an antibody-oligonucleotide probeconjugate, wherein the antibody molecule bears two or threeoligonucleotides, and then to add a specific volume, around 50, 75, 100,125, 150, 200, 225, 250, 300, or 400 microliters or less, of a solutionof a fluorescent dextran bearing a single complementary oligonucleotide.In certain embodiments, the concentrations of the first oligonucleotideand the second complementary oligonucleotide in the final solution wouldbe approximately equal based on moles of bases that are available tobase pair, allowing nearly complete formation of double strandedhybridization products. The resulting hybrid would thus comprise asingle antibody linked to one, two or three dextran scaffolds, where thelinker is comprised of a double stranded nucleic acid. Alternatively, incertain embodiments, a hybrid might contain a greater number ofdetectors linked to each probe, thereby achieving greater sensitivity.In certain embodiments, an antibody linked to one or moreoligonucleotides would be combined with a detector comprised of aparticle or bead or surface coated with a complementary oligonucleotide.Here, the hybrid would comprise the particle or bead or surface coatedwith the probe, linked by double-stranded nucleic acids. In certainembodiments, an antibody is joined to a fluorescent dextran in solution,the resulting hybrid is functionally equivalent, or substantiallyequivalent, to an antibody which has been covalently labeled with afluorophore and may be used for biological tests in the same manner as alabeled or tagged antibody, as for direct labeling. In certainembodiments, the probe is allowed to bind to its target prior toformation of the hybrid. Here, in certain embodiments, an antigen isexposed to an antibody-oligonucleotide conjugate as a probe. Then, adetector, such as the complementary oligonucleotide covalently linked toa fluorescent dextran or a fluorescent protein or an enzyme would beintroduced. Herein, the annealing or hybridization of the complementarysequences would then bring the detector into proximity with the targetvia its interaction with the probe. In these embodiments, the hybrid isformed in situ, to perform indirect labeling. In certain embodiments,where the probe is an antibody conjugated to one or more than oneoligonucleotide, and the probe is allowed to contact the target, andthen, where the detector is a particle or bead or surface coated withthe complementary oligonucleotide, and the detector is combined with thetarget and probe, a hybrid can be formed to capture the target and probeonto the solid material, by hybridization and formation of doublestranded nucleic acid linkers between the probe and detector components.

The term “preassembly” or “preassembly hybridization” or preassembledhybrids” may refer to assembly via hybridization of oligonucleotidesequence containing components prior to contacting a sample or binding atarget. For example, a molecular probe and a detectable component may bepreassembled via hybridization prior to either the molecular probe, thedetectable component, or both, contacting the sample, or prior to themolecular probe recognizing or binding a target. In certain embodiments,a molecular probe and a universal adapter may be preassembled viahybridization prior to either the molecular probe, the universaladapter, or both, contacting the sample, or prior to the molecular proberecognizing or binding a target. In certain embodiments, a detectablecomponent and a universal adapter may be preassembled via hybridizationprior to either the detectable component, the universal adapter, orboth, contacting the sample, or prior to the molecular probe recognizingor binding a target. In certain embodiments, a molecular probe and adetectable component and a universal adapter may be preassembled viahybridization prior to either the molecular probe, the detectablecomponent, the universal adapter, or combinations thereof, contactingthe sample, or prior to the molecular probe recognizing or binding atarget.

The term “linkage” may refer to the connection between two molecules,for example, the connection between two modified molecules. The linkagemay be formed by the formation of a covalent bond. Suitable covalentlinkage may include, but is not limited to the formation of an amidebond, an oxime bond, a hydrazone bond, a triazole bond, a sulfide bond,an ether bond, an enol ether bond, an ester bond, or a disulfide bond.The hydrazone bond may be, for example, a bis-arylhydrazone bond. Thelinkage may provide a UV-traceable characteristic that may be used todetect or quantify the amount of conjugate formed.

The term “spacer group” may refer to a molecular moiety or molecularsegment that may join atoms, molecules, or functional groups togetherthrough chemical bonds. Suitable spacer groups may be of sufficientlength or size such that the steric hindrance or steric clashes betweenthe joined components may be minimized. In certain embodiments, amolecular probe, such as a biomolecule-oligonucleotide conjugate maycomprise a spacer group located between the biomolecule and theoligonucleotide. The spacer group may, for example, minimize sterichindrance between two or more oligonucleotides on a single biomolecule,such as an antibody; may minimize steric hindrance between a signalgenerating moiety conjugated to an oligonucleotide; and/or may minimizesteric hindrances between multiple signal generating moieties on asingle detectable component and the oligonucleotide on said detectablecomponent. The spacer group may increase the solubility of thedetectable component or the molecular probe; may reduce steric hindranceand thereby improve detection efficiency; may prevent unwantedinteractions by shielding the joined components; may provide a generaland significant lower non-specific background for the detection methodand/or system; may reduce steric hindrance and thereby increase thebinding affinity of the molecular probe and/or the binding moiety for aparticular target; may reduce steric hindrance and thereby decrease thelevel of the background and risk of false positive detection signals;may reduce steric hindrance and thereby increase the hybridization ofthe molecular probe with the detectable component and/or universaladapter; may prevent or minimize the reduction of signal that isgenerated from a detectable component when the detectable component isin close proximity to another detectable component, such as when onesignal generating moiety in close proximity to another signal generatingmoiety; or combinations thereof. The spacer may be stable tothermolysis, stable to hydrolysis, may be biocompatible, or combinationsthereof. Suitable spacer groups may include, but are not limited to anethyleneoxide moiety, a polymer formed from repeating—(—CH₂—CH₂O—)—moieties, such as polymerized ethylene oxide, for example, polyethyleneglycol (PEG); polyethylene oxide (PEO); 6-amino-hexanoic acid;succimidyl 4-(N-malemidomethyl)cylohexane-1-carboxylate (SMCC). Thespacer group may also be a homobifunctional spacer group, such asdivinyl sulfone (DVS), glutaric dialdehyde, hexane di-isocyanate,dimethylapimidate, 1,5-difluoro-2,4-dinitrobenzene. In certainembodiments, the spacer group may be a heterobifunctional spacer group,such as N-gamma-maleimidobytyroloxy succinimide ester (GMBS). The spacergroup may be a zero length spacer groups, such as1-ethyl-3-(3-dimethylaminopropyl)cabodiimide.

The term “complementary reactive groups” may represent those groupsthat, when reacted together, form a covalent linkage. For example, ahydrazino group may be complementary to a carbonyl derivative. Forexample, an oxyamino group may also be complementary to a carbonylderivative. For example, an amino reactive group may refer to moietiesthat may react directly with amine moieties forming amide bonds. Forexample, a thiol reactive group may refer to moieties that may reactdirectly with sulfhydryl groups forming stable sulfide bonds.

The term “derivative of a compound” may include, for example, a salt,ester, enol ether, enol ester, solvate or hydrate thereof that may beprepared. Salts may include, but are not limited to, pharmaceuticallyacceptable salts; amine salts; alkali metal salts, such as but notlimited to lithium, potassium and sodium; alkali earth metal salts, suchas but not limited to barium, calcium and magnesium; transition metalsalts, such as but not limited to nickel, zinc, copper, cobalt, andiron; and other metal salts, such as but not limited to sodium hydrogenphosphate and disodium phosphate; and also may include, but is notlimited to, salts of mineral acids, such as but not limited tohydrochlorides and sulfates; and salts of organic acids, such as but notlimited to acetates, lactates, malates, tartrates, citrates, ascorbates,succinates, butyrates, valerates and fumarates. For example, esters mayinclude, but are not limited to, alkyl, alkenyl, alkynyl, aryl,heteroaryl, aralkyl, heteroaralkyl, cycloalkyl and heterocyclyl estersof acidic groups, including, but not limited to, carboxylic acids,phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids andboronic acids. Enol ethers may include, but are not limited to,derivatives of formula C══C(OR) where R is hydrogen, alkyl, alkenyl,alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl orheterocyclyl. Enol esters may include, but are not limited to,derivatives of formula C══C(OC(O)R) where R is hydrogen, alkyl, alkenyl,alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl orheterocyclyl. Solvates and hydrates are complexes of a compound with oneor more solvent or water molecule, for example, 1 to about 100, 1 toabout 10, 1 to about 2, 3 or 4, solvent or water molecules.

The term “amino acid” may refer to α-amino acids which are racemic, orof either the D- or L-configuration. The designation “d” preceding anamino acid designation (e.g., dAla, dSer, dVal, etc.) refers to theD-isomer of the amino acid. The designation “dl” preceding an amino aciddesignation (e.g., dlPip) refers to a mixture of the L- and D-isomers ofthe amino acid.

The term “synthetic molecule” may refer to a small molecule or polymerthat is not naturally derived.

In certain embodiments, the compounds provided herein may contain chiralcenters. Such chiral centers may be of either the (R) or (S)configuration, or may be mixtures thereof. For example, the compoundsprovided herein may be enantiomerically pure, diastereomerically pure,or stereoisomerically pure. The compounds provided herein may also bestereoisomeric mixtures or diastereomeric mixtures. For example, in thecase of amino acid residues, each residue may be of either the L or Dform. The preferred configuration for naturally occurring amino acidresidues is L.

The term “oligonucleotide” or “oligonucleotide sequence” or “oligo” or“oligonucleotide-barcode tag” or “oligo-barcode tag” may refer to anucleic acid, including, but not limited to, a ribonucleic acid (RNA); adeoxyribonucleic acid (DNA); a mixed ribonucleotide-deoxyribonucleotide,i.e., the oligonucleotide may include ribose or deoxyribose sugars or amixture of both; and analogs thereof; of various lengths; includingchromosomes and genomic material, such as PCR products or sequencingreaction products, for example, DNA including double and single strandedforms. Single stranded forms of the oligonucleotides are also provided.Alternatively, the oligonucleotide may include other 5-carbon or6-carbon sugars, such as, for example, arabinose, xylose, glucose,galactose, or deoxy derivatives thereof or other mixtures of sugars. Incertain embodiments, the oligonucleotide may refer to nucleic acidmolecules of 2-2000 nucleosides in length. In certain embodiments, theoligonucleotide sequence and/or an oligonucleotide sequencecomplementary to the oligonucleotide sequence, may comprise a3′-oligonucleotide, a 5′-oligonucleotide, a phosphorothioate, an LNA, aPNA, a morpholino, other alternative backbones, or combinations orderivatives thereof. Suitable oligonucleotide may be composed ofnaturally occurring nucleosides adenosine, guanosine, cytidine,thymidine and uridine, modified nucleosides, substituted nucleosides orunsubstituted nucleosides, purine or pyrimidine base, or combinationsthereof. Such purine and pyrimidine bases include, but are not limitedto, natural purines and pyrimidines such as adenine, cytosine, thymine,guanine, uracil, or other purines and pyrimidines, such as isocytosine,6-methyluracil, 4,6-di-hydroxypyrimidine, hypoxanthine, xanthine,2,6-diaminopurine, 5-azacytosine, 5-methyl cystosine, and the like. Thenucleosides may also be unnatural nucleosides. The nucleosides may bejoined by naturally occurring phosphodiester linkages or modifiedlinkages. The nucleosides may also be joined by phosphorothioatelinkages or methylphosphonate linkages.

The term “nucleobase” may refer to a heterocyclic moiety that is foundin naturally occurring oligonucleotides, including ribonucleic acids(RNA) and deoxyribonucleic acids (DNA), and analogs thereof, includingdeaza analogs. The nucleobase may include, but is not limited to,cytosines, uracils, adenines, guanines and thymines, and analogs thereofincluding deaza analogs.

The term “nucleotide analog” may refer to a peptide nucleic acid (PNA)and/or locked nucleic acid (LNA).

The term “universal adapter” may refer to a substance that may becapable of linking, for example, by hybridization, a molecular probe toa detectable component. For example, a universal adapter may comprise atleast two oligonucleotide sequence segments. The universal adapter mayfurther comprise at least one polymer and/or at least one spacer group.A universal adapter comprising the at least two oligonucleotide sequencesegments, may, for example, specifically hybridize a firstoligonucleotide sequence segment of the at least two oligonucleotidesequence segments to a molecular probe, and specifically hybridize asecond oligonucleotide sequence segment of the at least twooligonucleotide sequence segments to a detectable component. A universaladapter may comprise more than two oligonucleotide sequence segments,for example, multiple segments of the same oligonucleotide sequence ormultiple different oligonucleotide sequences. The universal adapter mayfurther be used to link one type of molecular probe to one or more thanone different detectable components, or vice versa. The universaladapter may also function as “master template” to link a molecular probeto several different detectable components. A universal adapter may linkdetectable component to one or more different kinds of molecular probes.A universal adapter may enhance and/or increase the signal generated,and subsequently detected, from a hybridized molecular probe comprisinga bound target and one or more detectable components, one or more signalgenerating moieties, and/or combinations thereof. In certainembodiments, the universal adapter comprising one or more of the sameoligonucleotide sequence segments may specifically hybridize to one ormore detectable components, which may increase the number of signalgenerating moieties linked to a given molecular probe bound to a targetin a sample, wherein the molecular probe is hybridized to said universaladapter.

The term “detectable component” may refer to a molecule comprising oneor more signal generating moieties and at least one oligonucleotidesequence that allow for the detection of the presence of a target, suchas a biological target, in a sample, in certain embodiments. The atleast one oligonucleotide sequence may be capable of linking or binding,such as by hybridization, directly to a molecular probe or indirectly toa molecular probe through an optional universal adaptor. The detectablecomponent may comprise a signal generating moiety conjugated directly toan oligonucleotide sequence. The detectable component may comprise oneor more signal generating moieties and an oligonucleotide sequence, forexample, one or more signal generating moieties conjugated directly toan oligonucleotide sequence. The detectable component may comprise oneor more signal generating moieties and an oligonucleotide sequence, forexample, one or more signal generating moieties conjugated indirectly toan oligonucleotide sequence. The detectable component may comprise oneor more signal generating moieties, an oligonucleotide sequence, and ascaffold, for example, one or more signal generating moieties conjugateddirectly scaffold comprising an oligonucleotide sequence, for example, ascaffold conjugated to an oligonucleotide sequence. For example, theoligonucleotide sequence may be conjugated directly to a scaffold, forexample a dextran or another hydrophilic polymer or a dendrimer,comprising one or more signal generating moieties. The oligonucleotidesequence of the detectable component may be a complementaryoligonucleotide sequence, for example, the oligonucleotide sequence ofthe detectable component may be complementary to an oligonucleotidesequence of a molecular probe.

The term “signal generating moiety” may refer to a molecule which may bedetected directly or indirectly so as to reveal the presence of a targetin the sample. In certain embodiments, a signal generating moiety may be“directly detectable” such that it may be detected without the need ofan additional molecule; for example, a directly detectable signalgenerating moiety may be a fluorescent dye, a luminescent species, aphosphorescent species, a radioactive substance, a nanoparticle, adiffracting particle, a raman particle, a metal particle, a magneticparticle, a bead, an RFID tag, or a microbarcode particle or othercombinations thereof. In certain embodiments, a signal generating moietymay be “indirectly detectable” such that it may require the employmentof one or more additional molecules to be detected; for example, anindirectly detectable signal generating moiety may be an enzyme thateffects a color change in a suitable substrate, as well as othermolecules that may be specifically recognized by another substancecarrying a label or react with a substance carrying a label, anantibody, an antigen, a nucleic acid or nucleic acid analog, a ligand, asubstrate, or a hapten. In certain embodiments, a signal generatingmoiety may be a fluorophore, sometimes called a fluorochrome (afluorescent compound); chromophores; biofluorescent proteins, such asphycoerythrin (R-PE), allophycocyanin (APC) and Peridinin ChlorophyllProtein Complex (PerCP); fluorophore labeled DNA dendrimers; QuantumDots or other fluorescent crystalline nanoparticles; tandem dyes, suchas a FRET dye; a chemiluminescent compound, a electrochemiluminescentlabel, a bioluminescent label, a polymer; a polymer particle; a bead orother solid surface; a Raman particle; a heavy metal chelate; gold orother metal particles or heavy atoms; a spin label; a radioactiveisotope; a secondary reporter; a hapten; aminohexyl; pyrene; a nucleicacid or nucleic acid analog; a protein; a peptide ligand or substrate; areceptor; an enzyme; an enzyme that catalyzes a color change in asubstrate; an enzyme substrate; an antibody; an antibody fragment; anantigen; or combinations or derivatives thereof.

The term “scaffold” may comprise a polymer, such as a hydrophilicpolymer, a biopolymer or a biologically inspired polymer, for example,an acrylate polymer; a substituted polyether; a substituted polystyrene;a polyethylene oxide; a nucleic acid; a polysaccharide molecule, such asa dextran; a linear polymer; a branched polymer; a dendrimer; orcombinations or derivatives thereof. The scaffold, such as a dendrimer,may be labeled by standard techniques, for example, by the use offluorochromes (or fluorescent compounds), enzymes (e.g., alkalinephosphatase and horseradish peroxidase), heavy metal chelates, secondaryreporters or radioactive isotopes.

The term “sample” may refer to a composition potentially containing atarget, such as a biological target.

The term “complex sample” may refer to a sample of material to beanalyzed that has multiple targets. For example, the sample may containat least 2, 5, 10, 15, 20, 30, 50, 75, 100, 500, 1000, 5000, 10,000,50,000, or 100,000 targets. In certain embodiments, the range of targetsmay be between 5 to 50, to 100, 25 to 100, 50 to 250, 50 to 5000, 500 to10,000, 250 to 50,000, 50 to 100,000, 15 to 500, 15 to 1000, 15 to10,000, or 20 to 10,000. This sample could be heterogeneous orhomogeneous mixture. The complex sample may also include those describedin the term “sample” provided herein.

The terms “targets” or “biological targets” may refer to one or moresubstances potentially present in a sample that are capable ofdetection.

The term “detection assay” or “detection method” or “method ofdetection” or “method for detection” may refer to a singleplex detectionassay or multiplex detection assay.

Molecular Probes, Antibodies and/or Biomolecule-OligonucleotideConjugates

A suitable molecular probe may comprise, for example, a monoclonalantibody, polyclonal antibody, antibody fragment, or a protein fragment,may be conjugated to an oligonucleotide, which may be detected by adetectable component, comprising a complementary oligonucleotidesequence and one or more signal generating moieties, by hybridizing theoligonucleotide of the molecular probe to the complementaryoligonucleotide sequence of the detectable component. In certainembodiments, the complementary oligonucleotide may be conjugated to oneor more signal generating moieties. The complementary oligonucleotidemay be conjugated to a scaffold, such as a dendrimer or a dextran,comprising the one or more signal generating moieties, such asfluorophors, secondary reporters, for example, a biotin, an enzyme, aheavy metal chelate, or a radioactive isotope, wherein the one or moresignal generating moieties may be detected. The one or more signalgenerating moieties may comprise combinations of different signalgenerating moieties. In certain embodiments, the molecular probe maycomprise a binding moiety conjugated to a hapten, wherein the hapten isfurther bound to a receptor-oligonucleotide conjugate, for example,antibody-biotin conjugate, wherein the biotin is further bound to aStreptavidin-oligonucleotide conjugate, or for example, anantibody-peptide conjugate, wherein the peptide is further bound to ananti-peptide-antibody-oligonucleotide conjugate.

A suitable molecular probe may comprise a binding moiety and anoligonucleotide, for example, a biomolecule-oligonucleotide conjugate,such as an antibody-oligonucleotide conjugate, an (antibodyfragment)-oligonucleotide conjugate, a protein-oligonucleotideconjugate, a (protein fragment)-oligonucleotide conjugate, apeptide-oligonucleotide conjugate, may have a molecular weight ofbetween about 15,000 Daltons and about 450,000 Daltons. For example, themolecular probe, may have a molecular weight of between about 25,000 andabout 400,000 Daltons, about 30,000 and about 350,000 Daltons; about15,000 and about 300,000 Daltons; 50,000 and about 250,000 Daltons;about 50,000 and about 200,000 Daltons; about 15,000 and about 75,000Daltons; or about 15,000 and about 50,000 Daltons. The molecular probe,may have a molecular weight of less than or about 450,000 Daltons,400,000 Daltons, 350,000 Daltons, 300,000 Daltons, 275,000 Daltons,225,000 Daltons, 200,000 Daltons, 175,000 Daltons, 150,000 Daltons,120,000 Daltons, 100,000 Daltons, 80,000 Daltons, 60,000 Daltons, 50,000Dalton, 40,000 Daltons, 30,000 Daltons; or 20,000 Daltons. The molecularweight of the molecular probe may affect the specific binding affinityto a target.

As used herein “specific binding” or “specifically binding” or “bindingaffinity” in certain embodiments may mean having a binding affinity asmeasured by dissociation constant for a specific target at less than10⁻⁴ molar (M), 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹² M, or10⁻¹⁵ M.

As used herein the amount of false positives generated by the detectionmethods may mean, in certain embodiments, events wherein the bindingmoiety binds to an unintended target in addition to binding to thedesired target. Similarly, as used herein the amount of false negativesgenerated by the detection methods may mean, in certain embodiments,events wherein the binding moiety binds to an unintended target at theexpense of binding to the desired target. For example, unintendedbinding of aggregates, debris, contaminants, plastic, glass or metalcontact surfaces, particles, containers, tubes, filters, and/or pippettetips. Another example would be an antibody binding to an antigen forwhich it has no binding affinity or substantially no binding affinity.In certain embodiments, the amount of false positives may be less than10%, 7%, 5%, 3% or 1% of the true positives. In certain embodiments, theamount of false positives generated by the detection methods may be lessthan those of secondary antibody detection methods. In certainembodiments, the amount of false negatives may be less than 10%, 7%, 5%,3% or 1% of the true positives. In certain embodiments, the amount offalse negatives generated by the detection methods may be less thanthose of secondary antibody detection methods.

As used herein the solubility of a molecular probe, comprising a bindingmoiety conjugated to one or more oligonucleotides, in certainembodiments may mean a molecular probe having solubility greater than,the same solubility, substantially the same solubility, or at least 98%,95%, 93%, 90%, 85%, 75%, 65%, or 50% of the solubility of theunconjugated binding moiety. In certain embodiments, the solubility ofthe molecular probe may be sufficient to minimize the non-specificbinding to a target. The solubility of the molecular probe may affectthe specific binding affinity to a target. For example, to one or morebiological targets.

As used herein neutral charge in certain embodiments may mean whereinthe solubility of a molecular probe does not need to be enhanced by theaddition of a polycharged species. For example, neutral charge may meanwherein the molecular probe is sufficiently soluble such that furthermodification of the molecular probe to enhance its solubility isunnecessary. In certain embodiments, neutral charge may mean wherein themolecular probe is sufficiently soluble to be utilized to be provided tothe sample such that further modification of the molecular probe toenhance its solubility is unnecessary.

A suitable antibody or immunoglobulin may comprise, for example, naturalantibodies, artificial antibodies, genetically engineered antibodies,monovalent antibodies, polyvalent antibodies, monoclonal antibodies,polyclonal antibodies, camelids, monobodies, scFvs and/or fragments orderivatives thereof. In certain applications, the antibody orimmunoglobulin molecules may be monoclonal, polyclonal, monospecific,polyspecific, humanized, single-chain, chimeric, camelid single domain,shark single domain, synthetic, recombinant, hybrid, mutated,CDR-grafted antibodies, and/or fragments or derivatives thereof. Incertain embodiments, antibodies may be derived from mammal species, forexample, rat, mouse, goat, guinea pig, donkey, rabbit, horse, lama,camel, or avian species, such as chicken or duck. The origin of theantibody is defined by the genomic sequence irrespective of the methodof production. The antibodies may be of various isotypes, e.g., IgG,IgM, IgA, IgD, IgE or subclasses, e.g., IgG1, IgG2, IgG3, IgG4. Theantibodies may be produced recombinantly, or by other means, which mayinclude antibody fragments which can still bind antigen, for example, anFab, an F(ab)₂, Fv, scFv, VhH, and/or V-NAR. The antibody, including anantibody fragment, may be recombinantly engineered to include anepitope, for example, a peptide. In certain embodiments, the epitope maybe a Myc tag, a FLAG tag, an HA tag, an S tag, a Streptag, an His tag, aV5 tag. In certain embodiments, the peptide tag may serve as a FlAshtag, a biotinylation tag, Sfp tag, or other peptide subject to covalentmodification. The antibody may be chemically modified to include ahapten, for example a small molecule or a peptide. The hapten may be anitrophenyl group, a dinitrophenyl group, a digoxygenin, a biotin, a Myctag, a FLAG tag, an HA tag, an S tag, a Streptag, a His tag, a V5 tag, aReAsh tag, a FlAsh tag, a biotinylation tag, Sfp tag, or other chemicalor peptide tag subject to covalent modification. Inclusion of an epitopeor hapten in an antibody or antibody fragment may facilitate subsequentbinding of a molecular probe, detectable component, binding moiety, orsignal generating moiety. In certain embodiments, peptide tag haptenschemically conjugated to protein binders can be used in conjunction withanti-peptide tag antibody-signal detector conjugates in singleplex andmultiplex immundetection assays, such as immunohistochemistry (IHC),flow cytometry, microscopy, imaging, high content screening (HCS),immunocytochemistry (ICC), immunomagnetic cellular depletion,immunomagnetic cell capture, in situ hybridization (ISH), enzymeimmuno-assay (EIA), enzyme linked immuno-assay (ELISA), ELISpot, arraysincluding bead arrays, multiplex bead array, microarray, antibody array,cellular array, solution phase capture, chemiluminescence detection,infrared detection, blotting method, a Western blot, a Southern blot, aSouthwestern blot, labeling inside an electrophoresis system, labelingon a surface, labeling on an array, PCR amplification, elongationfollowed by PCR amplification, immunoprecipitation,co-immunoprecipitation, chromatin immunoprecipitation, pretargetingimaging, therapeutic agent, or combinations thereof. The antibody mayinclude, for example, hybrid antibodies having at least two antigen orepitope binding sites, single polypeptide chain antibodies, bispecificrecombinant antibodies (e.g. quadromes, triomes), interspecies hybridantibodies, and molecules that have been chemically modified and may beregarded as derivatives of such molecules and which may be preparedeither by methods of antibody production or by DNA recombination, usinghybridoma techniques or antibody engineering or synthetically orsemisynthetically.

A suitable polyclonal antibody may be produced through a variety ofmethods. For example, various animals may be immunized for this purposeby injecting them with an antigen, for example the target biologicalmolecule, or another molecule sharing an epitope of the targetbiological molecule. Such antigen molecules may be of natural origin orobtained by DNA recombination or synthetic methods, or fragments thereofand the desired polyclonal antibodies are obtained from the resultingsera and may be purified. Alternatively, intact cells that array thetarget biological molecule may be used. Various adjuvants may also beused for increasing the immune response to the administration ofantigen, depending on the animal selected for immunization. Examples ofthese adjuvants include Freund's adjuvant, mineral gels such as aluminumhydroxide, surfactant substances such as polyanions, peptides, oilemulsions, haemocyanins, dinitrophenol or lysolecithin.

A suitable primary antibody may contain an antigen binding region whichcan specifically bind to an antigen target in a sample, such as animmunohistochemistry sample, may be employed. For example, a primaryantibody may be comprised within a primary binding moiety or a primarymolecular probe. A suitable secondary antibody may contain an antigenbinding region which can specifically bind to the primary antibody, forexample, the constant region of the primary antibody. The secondaryantibody may be conjugated to a polymer. The polymer may be conjugatedwith between about 2-20 secondary antibodies, or may be conjugated withbetween about 1-5 tertiary antibodies, such as 1, 2, 3, 4, or 5 tertiaryantibodies. The secondary antibody may act as a secondary bindingmoiety, while in other embodiments, the secondary antibody may act asmolecular probe, recognizing the target, such as an antigen, indirectlythrough a primary antibody. A suitable tertiary antibody may contain anantigen binding region which can specifically bind to the secondaryantibody, for example, a constant region of the secondary antibody, or ahapten linked to the secondary antibody or a polymer conjugated to thesecondary antibody. For example, the tertiary antibody may be conjugatedto a polymer, such as between about 1-20 tertiary antibodies. Thepolymer may be conjugated with between about 1-5 tertiary antibodies,such as 1, 2, 3, 4, or 5 tertiary antibodies. The tertiary antibody mayact as a tertiary binding moiety. In other embodiments, the tertiaryantibody may act as molecular probe, recognizing the target, such as anantigen, indirectly through a primary antibody and a secondary antibody.

The stoichiometry of the conjugation reaction to form thebiomolecule-oligonucleotide conjugates, for example, theantibody-oligonucleotide conjugates, protein-oligonucleotide conjugates,(protein fragment)-oligonucleotide conjugates, orpeptide-oligonucleotide conjugates, may comprise one equivalent ofmodified biomolecule and at least 0.5 equivalents of modifiedoligonucleotide. Other examples are at least 1.0 equivalent, at least1.5 equivalents, at least 2.0 equivalents, at least 2.5 equivalents, atleast 3.0 equivalents, at least 3.5 equivalents, or at least 4.0equivalents of modified oligonucleotide. The stoichiometry of theconjugation reaction to form the biomolecule-oligonucleotide conjugates,may comprise one equivalent of modified biomolecule and between about0.5 and about 2.0 of modified oligonucleotide, for example, betweenabout 1.5 and about 2.5 equivalents, between about 2.0 and about 2.5equivalents, between about 2.0 and about 3.0 equivalents, between about2.5 and about 3.5 equivalents, between about 3.0 and about 3.5equivalents, between about 3.0 and about 4.0 equivalents, or betweenabout 3.5 and about 4.5 equivalents modified oligonucleotide. In certainembodiments, the stoichiometry of the conjugation reaction may beadjusted to form biomolecule-oligonucleotide conjugates, for example,antibody-oligonucleotide conjugates, protein-oligonucleotide conjugates,or peptide-oligonucleotide conjugates, that retain sufficientimmunoreactivity of the biomolecule, such as an antibody, that has beenconjugated.

Suitable molecular probes, such as biomolecule-oligonucleotideconjugates, for example, antibody-oligonucleotide conjugates,protein-oligonucleotide conjugates, (protein fragment)-oligonucleotideconjugates, or peptide-oligonucleotide conjugates, may be theconjugation product of one modified biomolecule and on average between1.0 and 2.0 modified oligonucleotides that have conjugated to themodified biomolecule. For example, the biomolecule-oligonucleotideconjugates may be the conjugation product of one modified biomoleculeand on average between 1.0 and 2.0, between 1.5 and 2.5, between 2.0 and2.5, between 2.0 and 3.0, between 2.5 and 3.5, between 2.5 and 3.0,between 3.0 and 4.0, between 3.0 and 3.5, or between 3.5 and 4.5modified oligonucleotides that have conjugated to the modifiedbiomolecule.

Suitable molecular probes, such as biomolecule-oligonucleotideconjugates, for example, antibody-oligonucleotide conjugates,protein-oligonucleotide conjugates, (protein fragment)-oligonucleotideconjugates, or peptide-oligonucleotide conjugates, may comprise onaverage a molar ratio of about 1:1 to about 1:4.5, about 1:1 to about1:4, about 1:1 to about 1:3.5, about 1:1 to about 1:3, about 1:1 toabout 1:2.5, about 1:1 to about 1:2 or about 1:1 to about 1:1.5biomolecule to oligonucleotides. In certain embodiments, the molecularprobes may comprise on average a molar ratio of about 1:1, 1:2, 1:3, or1:4 biomolecule to oligonucleotides.

A suitable molecular probe may comprise a binding specificity for ananalyte, such as a target and/or biological target, for example, abinding specificity of about 10⁻⁴ M to about 10⁻¹⁵ M, about 10⁻⁵ M toabout 10⁻¹⁵ M, about 10⁻⁶ M to about 10⁻¹⁵ M, about 10⁻⁷ M to about10⁻¹⁵ M, about 10⁻⁹ M to about 10⁻¹⁵ M, or about 10⁻¹² M to about 10⁻¹⁵M for an analyte.

The biomolecule-oligonucleotide conjugates may be a mixture ofbiomolecule-oligonucleotide conjugates having modified oligonucleotidesthat have been conjugated to the modified biomolecule, but wherein thelinkage points of the oligonucleotides to the biomolecule are notuniformly identical across the entire sample. For example, a prepared,purified and isolated biomolecule-oligonucleotide conjugates sample mayhave one biomolecule-oligonucleotide conjugate that has one set oflinkage points for each of the oligonucleotides conjugated to thebiomolecule, and the same sample may have a differentbiomolecule-oligonucleotide conjugate that has a similar number ofoligonucleotides conjugated to that biomolecule, but having a differentset of linkage points for each of those oligonucleotides conjugated.

Suitable molecular probes may specifically bind to a molecule expressedby diseased cells and another molecular probe may specifically bind toanother molecule expressed by disease cells. For example, suchembodiments may be useful for diagnosing a disease in a subject wherethe disease may be better diagnosed by detecting a combination of two ormore markers in a sample. In these embodiments, one or more molecularprobes that specifically bind to a cell type specific marker also can beutilized. The sample may be from various sources, and may be aparticular set of cells or group of cells from a subject or patient.

Suitable molecular probes may specifically bind to a molecule expressedby a particular organism and another molecular probe may specificallybind to another molecule expressed by the organism. These embodimentsmay be useful for detecting an organism in a sample where the organismmay be better detected by identifying two or more markers in a sample.In certain embodiments, the one or more molecular probes mayspecifically bind to a cell type specific marker. Such embodiments maybe useful for detecting a particular strain of organism in a sample(e.g., a biological sample, a sample from animal meat for humanconsumption, or an environmental sample), where the strain isspecifically detected by a combination of a genus-associated moleculeand a species-associated molecule, for example. Such embodiments may beuseful for detecting a pathogenic organism in a biological sample fordiagnosing a disease caused by the organism (e.g., hepatitis C infectionin a human blood sample), and for detecting a particular organism in anenvironmental sample for agricultural and anti-bioterrorismapplications. For example, a molecular probe may be used to detect thepresence, absence or levels of beneficial bacteria in soil to determinesuitability for growing crops, and for detecting a pathogenic organismsuch as anthrax in soil or water samples for combating bioterrorism.

Suitable modified biomolecules may be prepared by reaction of abiomolecule of interest with one of the functionalities of abifunctional reagent. The modified biomolecules are then available forconjugation or immobilization using the remaining functional group. Incertain embodiments, the modified biomolecule may comprise one or moreof the following, comprising a modified protein; a modified peptide; amodified antibody; a modified glycoprotein; a modified monosaccharide; amodified disaccharide; a modified trisaccharide; a modifiedpolysaccharide; a modified dextran; a modified drug-like molecule; amodified drug; a modified small molecule; a modified pathogen; amodified toxin; a modified polymer; a modified biopolymer; a modifieddendrimer; a modified nuclear receptor; a modified nuclear receptorligand; a modified cytokine; a modified epitope; a modified peptideepitope; a modified antigen epitope; a modified pathogen epitope; amodified enzyme; a modified enzyme substrate; a modified cell-membraneprotein; and/or combinations or derivatives thereof.

As used herein the efficiency of the hybridization of a conjugatedoligonucleotide sequence to a conjugated complementary oligonucleotidesequence in certain embodiments may mean a conjugated oligonucleotidesequence and conjugated complementary oligonucleotide sequence having ahybridization efficiency of at least 98%, 95%, 93%, 90%, 85%, 75%, 65%,or 50% of the hybridization efficiency of the unconjugatedoligonucleotide. In certain embodiments, hybridization of at least 98%of a conjugated oligonucleotide sequence to a conjugated complementaryoligonucleotide sequence may be obtained by equimolar concentration ofthe conjugated complementary oligonucleotide sequence. In someembodiments, hybridization of at least 98% of a conjugatedoligonucleotide sequence to a conjugated complementary oligonucleotidesequence may be obtained by providing an excess of the conjugatedcomplementary oligonucleotide sequence, one and one tenth fold, one andone half fold, two fold, five fold, ten fold, one hundred fold, or onethousand fold.

Detection—Multiplex, Signal Generating Moiety

The oligonucleotide sequence on the detectable component may be aunique, distinguishable, and/or specifically designed oligonucleotidesequence complementary to the oligonucleotide sequence of the selectedmolecular probe. The oligonucleotide sequence on the molecular probe maybe a unique, distinguishable, and/or specifically designedoligonucleotide sequence complementary to the oligonucleotide sequenceof the selected detectable component. For example, a sample having afirst and a second target may be detected by a first molecular probebinding to a first target that is specifically hybridized with a firstdetectable component having a specifically designed complementaryoligonucleotide sequence to the first molecular probe, and a secondmolecular probe binding to a second target is specifically hybridizedwith a second detectable component having a specifically designedcomplementary oligonucleotide sequence to the second molecular probe.This flexibility to design the oligonucleotide sequences of themolecular probes and the detectable components permits the detection ofmultiple targets in a sample. For example, this permits the specificdesign of a multi-plex detection system wherein the target-boundmolecular probe may be detected with a choice of a great number ofsignal generating moieties, as compared to a directly labeled bindingmoiety, such as a labeled secondary antibody. This flexibility permitsthe specific design of a multi-plex detection system wherein the bindingaffinity of the molecular probe for the particular target is maintainedand unperturbed, as compared to a directly labeled binding moiety, suchas a labeled secondary antibody, which may be altered by the presence ofone or more signal generating moieties.

Enhanced signal, in certain embodiments, may mean wherein theenhancement of the signal may be related to the structure and nature ofthe detectable component, such as the structure and nature of thescaffold conjugated to the oligonucleotide. For example, the enhancementof the signal may be related to the number of signal generatingmoieties. The one or more detectable components may provide an enhancedsignal that minimizes detection errors from background noise. The one ormore signal generating moieties may provide an enhanced signal thatminimizes detection errors from background noise. In certainembodiments, the enhanced signal may be at least twice the signal of adetectable component comprising a single signal generating moietyconjugated to an oligonucleotide, such as at least 3×, 4×, 5×, 7×, 9×,or 10×. In certain embodiments, the amount of enhanced signal may behigher, for example, at least 20×, 30×, 50×, 100×, 500×, 1000×, 10,000×,and 100,000× the signal of a detectable component comprising a singlesignal generating moiety conjugated to an oligonucleotide. By enhancingthe signal this may have the advantage of reducing or further minimizingdetection errors from background noise. For example, in certainembodiments, the detections errors may be reduced or further minimizedby at least 5%, 7%, 9%, 10%, or 15%.

A suitable detectable component may comprise one or more universaladapters; may comprise at least one polymer and/or at least one spacergroups; or combinations thereof. In certain embodiments, the detectablecomponent may be used to detect one or more targets, at least twotargets, at least 3 targets; at least 4; at least 5; at least 10; atleast 15; at least 20; at least 25; at least 30; at least 35; at least40; at least 45; at least 50; at least 75; at least 100; at least 125;at least 150; at least 200; at least 400; at least 1,000; at least4,000; at least 10,000; or detect at least 50,000 targets within asample. In certain embodiments, multiple targets in a sample may not beexpressed in equal amounts, which may require differentialamplification.

A suitable detectable component may comprise one or more signalgenerating moieties, for example, a detectable component may comprise anaverage of between about 1 to about 100,000, about 1 to about 10,000,about 1 to about 1,000, about 1 to about 500, about 1 to about 100,about 1 to about 50, about 1 to about 20, about 1 to about 10, about 1to about 5, about 5 to about 50,000, about 5 to about 5,000, about 5 toabout 1,000, about 5 to about 100, about 5 to about 50, about 5 to about20; or about 5 to about 10 signal generating moieties.

The stoichiometry of the conjugation reaction to form the detectablecomponents, for example, a complementary oligonucleotide-signalgenerating moiety conjugate, or a complementary oligonucleotide-scaffoldconjugate, wherein the scaffold comprises one or more signal generatingmoieties, may comprise one equivalent of modified signal generatingmoiety and at least 0.5 equivalents of modified complementaryoligonucleotide or may comprise one equivalent of modified scaffold,comprising one or more signal generating moieties, and at least 0.5equivalents of modified complementary oligonucleotide. For example, thestoichiometry of the conjugation reaction to form the detectablecomponents, comprising a complementary oligonucleotide-signal generatingmoiety conjugate, may comprise one equivalent of modified signalgenerating moiety and at least 1.0 equivalents, 1.5 equivalents, 2.0equivalents, 2.5 equivalents, 3.0 equivalents, 3.5 equivalents, or 4.0equivalents of modified complementary oligonucleotide. For example, thestoichiometry of the conjugation reaction to form the detectablecomponents, comprising a complementary oligonucleotide-scaffoldconjugate, wherein the scaffold comprises one or more signal generatingmoieties, may comprise one equivalent of modified scaffold and at least1.0 equivalents, 1.5 equivalents, 2.0 equivalents, 2.5 equivalents, 3.0equivalents, 3.5 equivalents, or 4.0 equivalents of modifiedcomplementary oligonucleotide.

Suitable detectable components, may comprise a molar ratio of about 1:1complementary oligonucleotide to signal generating moiety. For example,the detectable component, may comprise a molar ratio of about 1:1complementary oligonucleotide to scaffold, wherein the scaffold, such asdextran, comprises one or more signal generating moieties, such as oneor more fluorophors. In certain embodiments, the number of signalgenerating moieties may be adjusted depending on the length of thescaffold, such as dextran or dendrimer that is utilized. For example,longer scaffolds, such as longer dextrans, may be utilized to increasethe number of signal generating moieties on a detectable component. Incertain embodiments, adjusting the number of signal generating moieties,for example, increasing the number, may adjust the sensitivity ofdetection, such increase the sensitivity of detection.

In certain embodiments, one or more complementary detectors wherein thefluorophores are directly conjugated to the oligonucleotide. Thesemulti-fluor detectors may be prepared from an oligonucleotide of 15-70bases wherein 2-10 fluorophores are directly conjugated to 2-10 basesmodified with reactive linker groups, e.g., amino groups, attached tothe base wherein the hydrogen bonding of the base is substantial notaffected (or not affected), i.e., on its minor groove side, and thefluorophores are spaced apart such that FRET does not occur or isminimized. In certain embodiments, the number of bases may vary, forexample, 10 to 30, 15 to 20, 15 to 30, 20 to 60, 40 to 70, etc. Incertain embodiments, the number of fluorophores may vary, for example, 2to 5, 3 to 8, 2 to 8, 4 to 10, 5 to 9, etc. FIG. 78 schematicallypresents the (A) preparation of the multi-fluor oligonucleotide, (B) itshybridization to an antibody-complementary oligonucleotide conjugate and(C) it binding to an antigen on a cell membrane, according to certainembodiments. It is recognized that the order of assembly may be changedwherein the antibody-oligonucleotide conjugate is added to the cell,allowed to bind, washed and then the multi-fluor hybrid is added andallowed to hybridized. FIG. 77 is a schematic presentation, according tocertain embodiments, of the use of antibody-oligonucleotide conjugatesand complementary oligonucleotides to which fluorophores are directlyconjugated wherein the antibody-oligonucleotide conjugate is added tothe biological sample and allowed to bind and subsequently detected bythe addition of the complementary oligonucleotide to which fluorophoresare directly conjugated.

A suitable signal generating moiety may be detected by the presence of acolor, or a change in color in the sample. In certain embodiments, morethan one type of signal generating moiety may be used, for example, byattaching distinguishable signal generating moiety to a singledetectable component or by using more than one detectable component,each carrying a different and distinguishable signal generating moiety.

A suitable signal generating moiety may be a protein, such as an enzyme,for example, alkaline phosphatase (AP); Horseradish Peroxidase (HRP);beta-galactosidase (βGAL); glucose-6-phosphate dehydrogenase;beta-N-acetylglucosaminidase; beta-glucuronidase; invertase; XanthineOxidase; firefly luciferase; or glucose oxidase (GO). Substrates thatmay be used for horse radish peroxidase (HRP) may include3,3′-diaminobenzidine (DAB); diaminobenzidine with nickel enhancement;3-amino-9-ethylcarbazole (AEC); benzidine dihydrochloride (BDHC);Hanker-Yates reagent (HYR); lndophane blue (IB); tetramethylbenzidine(TMB); 4-chloro-1-naphtol (CN); α-naphtol pyronin (α-NP); o-dianisidine(OD); 5-bromo-4-chloro-3-indolylphosphate (BCIP); Nitro blue tetrazolium(NBT); 2-(p-iodophenyl)-3-p-nitrophenyl-5-phenyl tetrazolium chloride(INT); tetranitro blue tetrazolium (TNBT); orδ-bromo-chloro-5-indoxyl-beta-D-galactoside/ferro-ferricyanide(BCIG/FF). Substrates that may be used for Alkaline Phosphatase mayinclude Naphthol-AS-B 1-phosphate/fast red TR (NABP/FR);Naphthol-AS-MX-phosphate/fast red TR (NAMP/FR); Naphthol-AS-B1-phosphate/fast red TR (NABP/FR); Naphthol-AS-MX-phosphate/fast red TR(NAMP/FR); Naphthol-AS-B1-phosphate/new fuschin (NABP/NF);bromochloroindolyl phosphate/nitroblue tetrazolium (BCIP/NBT); or5-Bromo-4-chloro-3-indolyl-β-δ-galactopyranoside (BCIG).

Other suitable signal generating moieties may be a heavy metal chelate,for example, europium, lanthanum, yttrium, gold; a dendrimer of heavymetal chelates; gold particles; or coated gold particles, which may beconverted by silver stains. Other suitable signal generating moietiesmay be a stable isotope bound to a chelator, for example, apolymer-based heavy metal chelates conjugated to antibodies and/or otherbinders may be used to multiplex protein analysis using a techniquenamed CyTOF (CYtometry Time Of Flight). Heavy metal isotopes of Ru, Rh,Pd, Ag, In, La, Hf, Re, Ir, Pt, Au, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho,Er, Tm, Yb and/or Lu may be used. In certain embodiments, a signalgenerating moiety may be a radioactive isotope, for example, ¹²⁵I, ¹³¹I,³H, ¹⁴C, ³⁵S, a radioactive isotop cobalt; a radioactive isotope ofselenium; or a radioactive isotope of phosphorous. In certainembodiments, a signal generating moiety may be a secondary reporter, forexample, biotin, streptavidin, avidin, digoxigenin, dinitrophenyl. Incertain embodiments, the signal generating moiety, such as a hapten, maybe conjugated to a fluorophore, peptide, nitrotyrosine, biotin, avidin,strepavidin, 2,4-dinitrophenyl, digoxigenin, bromodeoxy uridine,sulfonate, acetylaminoflurene, mercury trintrophonol, or estradiol. Incertain embodiments, a signal generating moiety may be a polymerparticle; micro particle; a bead; a latex particle of polystyrene, PMMAor silica. In certain embodiments, a signal generating moiety may be aparticle embedded with specific isotopes of heavy metals in definedrelative abundance as a barcode. In certain embodiments, a signalgenerating moiety may be a particle embedded with fluorescent dyes, orpolymer micelles or capsules which may contain dyes, enzymes orsubstrates. In certain embodiments, a signal generating moiety may beluminol, isoluminol, acridinium esters, 1,2-dioxetanes,pyridopyridazines, and/or ruthenium derivatives.

For example, a signal generating moiety may be a fluorophore.Fluorescence generally refers to the physical process in which light isemitted from the compound after a short interval following absorption ofradiation. Generally, the emitted light is of lower energy and longerwavelength than that absorbed. In certain embodiments, the energy may betransferred from one fluorophore to another prior to emission of light.In certain embodiments, the fluorescence of the fluorophores used hereincan be detected using standard techniques to measure fluorescence. Thefluorophore may be, for example, fluorescein, or its derivatives, suchas fluorescein-5-isothiocyanate (FITC), 5-(and 6)-carboxyfluorescein, 5-or 6-carboxyfluorescein, 6-(fluorescein)-5-(and 6)-carboxamido hexanoicacid, fluorescein isothiocyanate; rhodamine, or its derivatives, such astetramethylrhodamine and tetramethylrhodamine-5-(and-6)-isothiocyanate(TRITC). In certain embodiments, the fluorophore may comprise coumarindyes, such as (diethyl-amino)coumarin or7-amino-4-methylcoumarin-3-acetic acid, succinimidyl ester (AMCA);sulforhodamine 101 sulfonyl chloride, TexasRed™, TexasRed™ sulfonylchloride; 5-(and-6)-carboxyrhodamine 101, succinimidyl ester, also knownas 5-(and-6)-carboxy-X-rhodamine, succinimidyl ester (CXR); lissamine orlissamine derivatives such as lissamine rhodamine B sulfonyl Chloride(LisR); 5-(and-6)-carboxyfluorescein, succinimidyl ester (CFI);fluorescein-5-isothiocyanate (FITC); 7-diethylaminocoumarin-3-carboxylicacid, succinimidyl ester (DECCA); 5-(and-6)-carboxytetramethylrhodamine,succinimidyl ester (CTMR); 7-hydroxycoumarin-3-carboxylic acid,succinimidyl ester (HCCA); 6-fluorescein-5-(and-6)-carboxamidolhexanoicacid (FCHA);N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacenepropionicacid, succinimidyl ester; also known as 5,7-dimethyl BODIPY™ propionicacid, succinimidyl ester (DMBP); “activated fluorescein derivative”(FAP), available from Molecular Probes, Inc.; eosin-5-isothiocyanate(EITC); erythrosin-5-isothiocyanate (ErITC); and Cascade™ Blueacetylazide (CBAA) (the O-acetylazide derivative of1-hydroxy-3,6,8-pyrenetrisulfonic acid). In certain embodiments, thefluorophore may comprise fluorescent proteins such as phycoerythrin,allophycocyanin, green fluorescent protein and its analogs orderivatives, fluorescent amino acids such as tyrosine and tryptophan andtheir analogs, fluorescent nucleosides, and other fluorescent moleculessuch as organic dyes, including Cy2, Cy3, Cy 3.5, Cy5, Cy5.5, Cy 7, IRdyes, Dyomics dyes, Oregon green 488, pacific blue, rhodamine green, andAlexa dyes. In certain embodiments, the fluorophore may take advantageof fluorescence energy transfer and comprise conjugates ofR-phycoerythrin or allophycocyanin to organic dyes, such as Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy 7, Dyomics dyes, or Alexa dyes. In certainembodiments, the fluorophore may comprise an inorganic fluorescentcolloidal particle such as a quantum dot or other fluorescentnanoparticle, such as particles based on semiconductor material likeCdS-coated CdSe nanocrystallites.

In certain embodiments, the signal generating moiety may be linked tothe oligonucleotide sequence by a covalent attachment or a non-covalentattachment. The signal generating moiety may also be linked to theoligonucleotide sequence before, during, or after the hybridizationevent.

In certain embodiments, a molecular probe, universal adaptor, and/ordetectable component may be pre-hybridized prior to bringing thecomposition into contact with a sample, comprising one or more moleculartargets. For example, three or more, four or more, five or moremolecular probes, universal adapters, and/or detectable components, maybe pre-hybridized prior to bringing the composition into contact with asample comprising one or more molecular targets. In certain embodiments,two or more molecular probes, universal adapters, and/or detectablecomponents, each of which may comprise one or more spacer groups, may bepre-hybridized prior to bringing the composition into contact with asample, comprising one or more molecular targets.

Samples and/or Targets

A suitable sample may comprise one or more targets, such as one or moreof a protein; a peptide; a carbohydrate; a nucleic acid; a lipid; asmall molecule; a toxin; a drug or drug-like molecule, or derivativesthereof; or may comprise a combination of targets that may be proteins;peptides; carbohydrates; nucleic acids; lipids; small molecules; toxins;drugs or drug-like molecules, or derivatives thereof. For example, asample may comprise a defined combination of natural and/or chemicallysynthesized species. In certain embodiments, the composition of a samplemay not be fully known. The sample may include a cell, a group of cells,may be prepared from a cell or group of cells, may be a purifiedfraction from a cell preparation, may be a purified molecule. Forexample, the sample may comprise cells, such as mammalian cells (e.g.,human cells); insect cells; yeast cells; fungal cells; and/or bacterialcells. The cells, for example, may be from multicellular organism (e.g.,insects and mammals) derived from specific portions of the organism(e.g., specific tissues, organs, or fluids). Cells may be contacted byhybrids in vitro or in vivo, and may be contacted by hybrids when insuspension or when attached to a solid surface. Cells may not besignificantly modified during the process, may be fixed to a solidsupport, and/or may be made permeable using standard methods. The samplemay include cells, products produced by cells, cellular components,and/or mixtures thereof. The sample may include cellular components,such as a nucleus, cytoplasm, plasma cell membrane, nucleolus,mitochondria, vacuoles, subcellular organelles, endoplasmic reticulumand/or Golgi apparatus. The sample may include cells, tissue samples,and/or organs, such as molecular antigens produced from groups of cells,tissue samples, and/or organs. In certain embodiments, the sample maycomprise or be derived from, but not limited to, clinical, industrial,agricultural and environmental samples. For example, sample materialoften may be of medical, veterinary, environmental, nutritional orindustrial significance, and include body fluids, such as blood, serum,plasma, cerebrospinal fluid, synovial fluid, saliva, milk, sputum, lungaspirates, mucus, teardrops, exudates, secretions, urine, and fecalmatter; microbial culture fluids; aerosols; crop materials; animal meat(e.g., for human consumption or animal feed); and soils and groundwaters. In certain embodiments, the sample may comprise, but is notlimited to, molecules in pathogens, viruses, bacteria, yeast, fungi,amoebae and insects; molecules in diseased or non-diseased pest animalssuch as mice and rats; molecules in diseased and non-diseased domesticanimals, such as domestic equines, bovines, porcines, caprines, canines,felines, avians and fish; and molecules in diseased and non-diseasedhumans. In certain embodiments, the sample may comprise, but is notlimited to, biological samples derived from a human or other animalsource (such as, for example, body fluids, such as blood, serum, plasma,cerebrospinal fluid, synovial fluid, saliva, milk, sputum, lungaspirates, mucus, teardrops, exudates, secretions, urine, a biopsysample, a histology tissue sample, a PAP smear, a mole, a wart, etc.)including samples derived from a bacterial or viral preparation, as wellas other samples (such as, for example, agricultural products, waste ordrinking water, milk or other processed foodstuff, air, etc.). Incertain embodiments, the sample may comprise one or more of thefollowing: tissue cells, cells cultured in vitro, recombinant cells,infected cells, cells from laboratory animals, cells from mammalpatients, cells from human patients, mesenchemal stem cells, stem cells,immuno-competent cells, adipose cells, fibroblasts, natural-killer cells(NK-cells), monocytes, lymphocytes, lymph node cells, T-cells, B-cells,exudate cells, effusion cells, cancer cells, blood cells, red bloodcells, leukocytes, white blood cells, organ cells, skin cells, livercells, splenocytes, kidney cells, intestinal cells, lung cells, heartcells, or neuronal cells.

Suitable samples may comprise a range of analytes, such as targetsand/or biological targets, having a wide range of binding specificities,for example, about 10⁻⁴ M to about 10⁻¹⁵ M, about 10⁻⁵ M to about 10⁻¹⁵M, about 10⁻⁶ M to about 10⁻¹⁵ M, about 10⁻⁷ M to about 10⁻¹⁵ M, about10⁻⁹ M to about 10⁻¹⁵ M, or about 10⁻¹² M to about 10⁻¹⁵M.

As used herein homogeneous in certain embodiments may mean a samplehaving substantially the same class of targets, or the same class oftargets, for example, at least 60% 70%, 80%, 90%, 95%, or 98% of thesame class of targets. For example, classes of targets may include, butare not limited to the class of proteins, the class of sugar-containingcompounds, the class of antibodies, the class of peptides, the class oftoxins, the class of pathogens, or the class of antigens.

A suitable target or biological target may include, but is not limitedto, an antigen; an antibody; an enzyme; an enzyme substrate; a nuclearreceptor; a nuclear receptor ligand; a co-factor; a pathogen; a toxin; aprotein, such as a glycoprotein, a lipoprotein, a phosphoprotein, anacetylated protein, an hydroxylated protein, a sulfonated protein, anitrosylated protein, or a methylated protein; a protein fragment, suchas a peptide, a polypeptide or a modified polypeptide; a nucleic acid,such as a nucleic acid molecule, a nucleic acid segment, a nucleic acidmolecule of a pathogen or tumor cell, a mutated nucleic acid, a variantnucleic acid, a modified nucleic acid, a methylated nucleic acid, or anoxidatively damaged nucleic acid; an epitope; a lipid; a glyco-lipid; asugar; a carbohydrate-containing molecule, such as disaccharide,oligosaccharide, or a polysaccharide; a starch; a drug or drug-likemolecule; a small molecule; a salt; an ion; or one of a variety of otherorganic and inorganic substances; which may be free in solution or boundto another substance; or combinations or derivatives thereof. The targetmay be recognized by, for example, a suitable molecular probe,comprising a binding moiety. The recognition may be direct, while inother embodiments, the recognition may be indirect, via another bindingmoiety, such as by at least one primary, secondary, or higher orderbinding moiety. The target may be expressed on the surface of thesample, such as on a membrane, cell-membrane, or interface. The targetmay be contained in the interior of the sample. In the case of a cellsample, for example, an interior target may comprise a target locatedwithin the cell membrane, periplasmic space, cytoplasm, or nucleus, orwithin an intracellular compartment or organelle. The target may includeviral particles, or portions thereof, for example, a nucleic acidsegment or a protein. The viral particle may be a free viral particle,i.e., not associated with another molecule, or it may be associated witha sample described above. The target may include products derived fromDNA damage, an infective agent (e.g., a virus, bacterium, or fungus), anucleotide analog or derivative (e.g., bromodeoxyuridine (BrdU)) or amodified nucleotide (e.g., a biotinylated nucleotide)); a small organicor inorganic compound; an antisense nucleic acid (e.g., a PNA); acatalytic nucleic acid (e.g., a ribozyme); an inhibitory nucleic acid(e.g., a short inhibitory RNA (siRNA)); a polypeptide (e.g a cytokine orgrowth factor); an antibody or a peptide mimetic. For example, smallorganic or inorganic compounds may have a molecular weight of 10,000g/mol or less, 5,000 g/mol or less, 1,000 g/mol or less, or 500 g/mol orless. Compounds may be obtained using combinatorial library methods,such as spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; “one-beadone-compound” library methods; and synthetic library methods usingaffinity chromatography selection. The libraries may include siRNAmolecule libraries or peptide mimetic libraries.

Other suitable targets may include a molecular antigen, which may be apeptide or protein or may comprise a portion of a peptide or protein.For example, the target may be an antigen, such as a subregion of aprotein, such as in the N-terminus, C-terminus, extracellular region,intracellular region, transmembrane region, active site (e.g.,nucleotide binding region or a substrate binding region), a domain(e.g., an SH2 or SH3 domain) or a post-translationally modified region(e.g., phosphorylated, glycosylated, methylated, acetylated,nitrosylated, sulfated, farnesylated, myristoylated, palitoylated,sumoylated or ubiquinylated region). The target may be an antigen,comprising a modification moiety or a portion thereof (e.g., theglycosyl group or a portion thereof) or may be a modification moiety inconjunction with amino acids of the protein or peptide to which it islinked (e.g., a phosphoryl group in combination with one or more aminoacids of the protein or peptide). The protein may be a signaltransduction factor, cell proliferation factor, apoptosis factor,angiogenesis factor, senescence factor, or cell interaction factor.Suitable examples of cell interaction factors may include, but are notlimited to, cadherins (e.g., cadherins E, N, BR, P, R, and M;desmocollins; desmogleins; and protocadherins); connexins; integrins;proteoglycans; immunoglobulins, cell adhesion molecules (e.g., ALCAM,NCAM-1 (CD56), ICAM-1 and ICAM-2, CD44, LFA-1, LFA-2, LFA-3, LECAM-1,VLA-4, ELAM and N-CAM); selectins (e.g., L-selectin (CD62L), E-selectin(CD62e), and P-selectin (CD62P)); agrin; CD34; and a cell surfaceprotein that is cyclically internalized or internalized in response toligand binding. Examples of signal transduction factors may include, butare not limited to, protein kinases (e.g., mitogen activated protein(MAP) kinase and protein kinases that directly or indirectlyphosphorylate it, Janus kinase (JAK1), cyclin dependent kinases,epidermal growth factor (EGF) receptor, platelet-derived growth factor(PDGF) receptor, fibroblast-derived growth factor receptor (FGF),insulin receptor and insulin-like growth factor (IGF) receptor); proteinphosphatases (e.g., PTPIB, PP2A and PP2C); GDP/GTP binding proteins(e.g., Ras, Raf, ARF, Ran and Rho); GTPase activating proteins (GAFs);guanine nucleotide exchange factors (GEFs); proteases (e.g., caspase 3,8 and 9), ubiquitin ligases (e.g., MDM2, an E3 ubiquitin ligase),acetylation and methylation proteins (e.g., p300/CBP, a histone acetyltransferase) and tumor suppressors (e.g., p53, which is activated byfactors such as oxygen tension, oncogene signaling, DNA damage andmetabolite depletion). The protein may be a nucleic acid-associatedprotein (e.g., histone, transcription factor, activator, repressor,co-regulator, polymerase or origin recognition complex (ORC) protein),which directly binds to a nucleic acid or binds to another protein boundto a nucleic acid.

Detection Assay

A suitable detection assay may comprise or may be used in connectionwith singleplex and multiplex assays, such as immunoassays, proteindetection assays, immunodetection, enzyme linked immuno-assays (ELISA),immunomagnetic cellular depletion, immunomagnetic cell capture, flowcytometry, immunohistochemistry (IHC), immunocytochemistry (ICC), insitu hybridization (ISH), ELISpot, enzyme immuno-assays (EIA), blottingmethods (e.g. Western, Southern, Southwestern, and Northern), arrays,bead arrays, multiplex bead array, microarray, antibody array, cellulararray, solution phase capture, chemiluminescence detection, infrareddetection, labeling inside electrophoresis systems or on surfaces orarrays, PCR amplification, elongation followed by PCR amplification,precipitation, immunoprecipitation, co-immunoprecipitation, chromatinimmunoprecipitation, pretargeting imaging, therapeutic agent, nucleicacid hybridization assays, microspcopy, imaging, high content screening(HCS), other assay or detection formats, for example, that are useful inresearch as well as in diagnosing diseases or conditions, orcombinations or derivatives thereof. In certain embodiments, the assaymay analyze expression patterns of genes or levels of proteins within asample. In certain embodiments, the IHC, ISH and cytological techniquesmay be performed in a matrix of tissue, cell and proteins which may bepartly cross-linked and very inhomogeneous in nature. In certainembodiments, the assay may be an IHC method of detecting targets usingeither direct labeling or secondary antibody-based or hapten-basedlabeling, such as EnVision™ (DakoCytomation), Powervision®(Immunovision, Springdale, Ariz.), the NBA™ kit (Zymed LaboratoriesInc., South San Francisco, Calif.), HistoFine® (Nichirei Corp, Tokyo,Japan). In certain embodiments, the methods disclosed herein may providean enhanced signal or an increased flexibility in IHC detectionplatforms.

Isolating Biomolecule-Oligonucleotide Conjugates and/or ModifiedOligonucleotide

In certain embodiments, methods for isolatingbiomolecule-oligonucleotide conjugates may comprise: i) introducing amodified biomolecule into a buffered solution; ii) conjugating themodified biomolecules with at least one modified oligonucleotide atgreater than about 80% efficiency to form biomolecule-oligonucleotideconjugates; and iii) isolating the biomolecule-oligonucleotideconjugates from the conjugation solution by binding the conjugates to animmobilized binder. The methods may comprise conjugation at greater thanabout 85%, greater than 90%, greater than 95%, or greater than about 98%efficiency to form biomolecule-oligonucleotide conjugates. In certainembodiments, other isolation techniques may be used, for example, sizeexclusion chromatography. In certain embodiments, the isolationtechnique may be selected from one or more of the following:chromatography, affinity chromatography, size exclusion chromatography,HPLC, reverse-phase chromatography, electrophoresis, capillaryelectrophoresis, polyacrylamide gel electrophoresis, agarose gelelectrophoresis, free flow electrophoresis, differential centrifugation,thin layer chromatography, immunoprecipitation, hybridization, solventextraction, dialysis, filtration, diafiltration, tangential flowfiltration, ion exchange chromatography, or hydrophobic interactionchromatography.

For example, the modified oligonucleotide may be prepared by reactingwith a bifunctional molecular reagent containing a first reactivecomponent that forms a covalent bond with the oligonucleotide, and asecond reactive component that may form a linkage with a complementaryreactive component on a modified biomolecule or a tagged biomolecule. Incertain embodiments, the second reactive component may be protected suchthat it will not react until removed following incorporation onto theoligonucleotide.

A suitable modified oligonucleotide may be prepared by incorporatingamino groups either 3′, 5′ or internally using other methods andreagents. For example, the modified oligonucleotide may be prepared byreacting with a moiety that is a bifunctional molecular reagent, such asan aromatic aldehyde or ketone, aromatic hydrazino or oxyaminomodification reagent, to incorporate a hydrazino or oxyamino functionrespectively.

A suitable modified oligonucleotide may be prepared bypost-synthetically modification of oligonucleotides prepared viapolymerases or reverse transcriptases with nucleoside triphosphatespossessing an aromatic aldehyde, aromatic hydrazine, oxyamino, or anamino group. For example, the modified oligonucleotide may be preparedby post-synthetically modification of oligonucleotides by incorporationof an aromatic aldehyde or ketone, aromatic hydrazino or oxyamino group,using a moiety that is a bifunctional molecular reagent, such as anaromatic aldehyde or ketone, aromatic hydrazino or oxyamino reagent.

The modified biomolecules, such as, modified antibodies, modifiedproteins, or modified peptides, may be prepared from biomolecules thatare derived from eukaryotic cells. The modified biomolecules may also beprepared from biomolecules that are derived from prokaryotic cells. Themodified biomolecule may include a molecular tag. The modifiedbiomolecule may be modified antibody, wherein the modified antibody maybe prepared from an antibody that contains a histidine rich sequencenear the hinge region. In certain embodiments, the modified biomoleculemay be modified antibody, wherein the modified antibody may be exclusiveof, i.e., do not contain, a histidine rich sequence near the hingeregion.

In certain embodiments, the phosphorus-containing moieties of themodified oligonucleotides may contain, for example, a phosphate,phosphonate, alkylphosphonate, aminoalkyl phosphonate, thiophosphonate,phosphoramidate, phosphorodiamidate, phosphorothioate,phosphorothionate, phosphorothiolate, phosphoramidothiolate, andphosphorimidate. The phosphorus-containing moieties of the modifiedoligonucleotides may be modified with a cationic, anionic, orzwitterionic moiety. The modified oligonucleotides may also containbackbone linkages which do not contain phosphorus, such as carbonates,carboxymethyl esters, acetamidates, carbamates, acetals, and the like orderivatives thereof.

A suitable modified biomolecule may be a modified antibody, comprisingan antibody that includes a histidine-rich region, for example, anantibody having a histidine-rich region near the hinge region of theantibody. The modified antibody may comprise an antibody that isexclusive of having a histidine-rich region. The modified antibody maycomprise an antibody that is of the IgG type antibody or the IgM typeantibody. The modified antibody may comprise one or more molecular tags,for example, but not limited to, a poly-histidine tag, a Flag Tag, a Myctag, or a peptide tag that an antibody has been raised against. Themodified antibody may comprise a poly-histidine fusion protein. Themodified antibody may comprise one or more spacer groups, for example,such as a polyethylene glycol (PEG) or a polyethylene oxide group (PEO).The modified antibody may comprise one or moieties that include areactive group, for example, a reactive group that may form a covalentbond when reacted with a complementary reactive group that may be partof a modified oligonucleotide. The modified antibody may be, forexample, a HyNic or 4FB-modified antibody.

A suitable modified biomolecule may be a modified protein or a modifiedpeptide, comprising a protein that includes a histidine-rich region, forexample, a protein having a histidine-rich region incorporated duringsolid phase synthesis. For example, the modified protein may comprise aprotein that is exclusive of having a histidine-rich region. In certainembodiments, the modified protein may comprise one or more moleculartags, for example, but not limited to, a poly-histidine tag, a Flag Tag,a Myc tag, or a peptide tag that an antibody has been raised against.The modified protein may comprise a poly-histidine fusion protein. Themodified protein may comprise one or more spacer groups, for example,such as a polyethylene glycol (PEG) or a polyethylene oxide group (PEO).The modified protein may comprise one or moieties that include areactive group, for example, a reactive group that may form a covalentbond when reacted with a complementary reactive group that may be partof a modified oligonucleotide. The modified protein may be, for example,a HyNic or 4FB-modified protein.

In certain embodiments, at least one modified oligonucleotide maycomprise one or more oligonucleotides that have been modified, forexample, at least two modified oligonucleotides, at least three, atleast four modified oligonucleotides. The at least one modifiedoligonucleotide may comprise two different modified oligonucleotides,for example, three different modified nucleotides or four differentmodified oligonucleotides. The at least one modified oligonucleotide maycomprise one or more spacer groups, for example, a PEG or PEO group. Themodified oligonucleotide may comprise one or moieties that include areactive group, for example, a reactive group that may form a covalentbond when reacted with a complementary reactive group that may be partof a modified antibody. The modified oligonucleotide may be, forexample, a 4-FB-modified oligonucleotide.

In certain embodiments, the biomolecule-oligonucleotide conjugates, forexample, antibody-oligonucleotide conjugates, protein-oligonucleotideconjugates, (protein fragment)-oligonucleotide conjugates, orpeptide-oligonucleotide conjugates, may be purified and/or isolated bybinding to an immobilized binder. A suitable immobilized binder maycomprise a metal ion, for example, a divalent metal ion, such as atransition metal ion. The metal ion may include, but is not limited to,a nickel ion, a zinc ion, a copper ion, an iron ion, or a cobalt ion.The metal ion may be immobilized by chelation to a stationary phase in acolumn. A suitable stationary phase may comprise an organic chelatorthat immobilizes and/or binds the metal ion. For example, the organicchelator may be selected from the group that includes, but is notlimited to, iminodiacetic acid, nitrilotriacetic acid, and/orbicinchoninic acid. The stationary phase may be a water insolublesupport, for example, the stationary phase may be agarose.

A suitable immobilized binder may comprise an immobilized antibody. Theimmobilized antibody may recognize and bind a portion of the modifiedbiomolecule, such as a modified antibody, and/or a portion of thebiomolecule-oligonucleotide conjugates, such as anantibody-oligonucleotide conjugate. The immobilized antibody mayrecognize and bind a modified biomolecule comprising a molecular tag,wherein the immobilized antibody is an antibody that has been raised toinclude that particular molecular tag. The immobilized antibody mayrecognize and bind the linkage formed during the conjugation reaction ofthe modified biomolecules, such as modified antibodies, modifiedproteins, or modified peptides, and the modified oligonucleotide,wherein the immobilized antibody is an antibody that has been raised toinclude that particular conjugation linkage.

Other suitable biomolecule-oligonucleotide conjugates, for example,antibody-oligonucleotide conjugates, protein-oligonucleotide conjugates,(protein fragment)-oligonucleotide conjugates, orpeptide-oligonucleotide conjugates, may be purified and/or isolated byadding the conjugation reaction mixture to a column having a stationaryphase comprising a binder that has been immobilized, or substantiallyimmobilized, to the stationary phase. The immobilized binder maycomprise an immobilized antibody bound to the stationary phase. Theimmobilized binder may comprise a metal ion, for example, a divalentmetal ion, such as a transition metal ion. The metal ion may beimmobilized by chelation to a stationary phase in a column. The metalion may include, but is not limited to, a nickel ion, a zinc ion, acopper ion, an iron ion, or a cobalt ion.

Preparing, Purifying, and/or Isolating the Biomolecule-OligonucleotideConjugates

A suitable method of preparing, purifying, and/or isolating thebiomolecule-oligonucleotide conjugates may be by selectively binding theconjugates to a binder that is immobilized, or substantiallyimmobilized, on a stationary phase, eluting the reaction components awayfrom the bound conjugate, and then releasing thebiomolecule-oligonucleotide conjugates by adding a displacing agent thatis selective for the immobilized binder. The method for isolatingbiomolecule-oligonucleotide conjugates, may comprise: i) conjugating amodified biomolecule with at least one modified oligonucleotide to formbiomolecule-oligonucleotide conjugates, wherein greater than 80% of themodified biomolecules are conjugated; ii) adding the conjugationreaction mixture to a column having a stationary phase comprising abinder that has been immobilized to the stationary phase; iii) bindingthe biomolecule-oligonucleotide conjugates selectively to theimmobilized binder; iv) eluting reaction components away from the boundbiomolecule-oligonucleotide conjugates; and v) isolating thebiomolecule-oligonucleotide conjugates by releasing the boundbiomolecule-oligonucleotide conjugates with a displacing agent selectivefor the binder. The immobilized binder may be a metal ion and thedisplacing agent may be a solution comprising a chelator for the metal,for example, EDTA. The immobilized binder may be an immobilized antibodyand the displacing agent may be a solution comprising a molecular tagthat is recognized by the immobilized antibody.

In certain embodiments, the method of preparing, purifying, and/orisolating the molecular probes, such as biomolecule-oligonucleotideconjugates, for example, antibody-oligonucleotide conjugates,protein-oligonucleotide conjugates, (protein fragment)-oligonucleotideconjugates, or peptide-oligonucleotide conjugates, may be mild, robust,simple, high yielding or combinations thereof. For example, the methodmay yield at least about 30% isolated molecular probes, for example,biomolecule-oligonucleotide conjugates, with respect to startingmodified biomolecule. In other methods, the yield may be at least 40%,50%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95% isolated molecularprobe, such as, biomolecule-oligonucleotide conjugates, with respect tostarting modified biomolecule. In other methods, the purity of theprepared, purified, and/or isolated molecular probe may be at least 40%,50%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%.

In certain embodiments, the method of preparing, purifying, and/orisolating the biomolecule-oligonucleotide conjugates may provide morethan one process by which to bind and release thebiomolecule-oligonucleotide conjugates. For example, the formedbiomolecule-oligonucleotide conjugates may be antibody-oligonucleotideconjugates, that may comprise a histindine-rich region included in thehinge region of the biomolecule, for example, antibody, which may bebound by chelating to a metal ion immobilized on a column, and theformed biomolecule-oligonucleotide conjugates, such asantibody-oligonucleotide conjugates, may further comprise a moleculartag that is recognized and may be bound by an antibody, for example, anantibody immobilized on a stationary phase. For example, the formedbiomolecule-oligonucleotide conjugates may be antibody-oligonucleotideconjugates, that may comprise a biomolecule, for example, an antibody,that is exclusive of, i.e., does not include a histidine-rich region,and the formed biomolecule-oligonucleotide conjugates, such asantibody-oligonucleotide conjugates, may further comprise a moleculartag that is recognized and may be bound by an antibody, for example, anantibody immobilized on a stationary phase, and wherein the moleculartag may also be bound by chelating to a metal ion. For example, themolecular tag may be a histidine-rich His-6 tag.

In certain embodiments, the biomolecule-oligonucleotide conjugates, forexample, antibody-oligonucleotide conjugates, protein-oligonucleotideconjugates, (protein fragment)-oligonucleotide conjugates, orpeptide-oligonucleotide conjugates, may comprise one or more detectablefluorophores, two or more detectable fluorophores, or three or moredetectable fluorophores. In certain embodiments, thebiomolecule-oligonucleotide conjugates may comprise two or moredifferent modified oligonucleotides, for example, three or moredifferent modified oligonucleotides, that have conjugated to thebiomolecule, where in each modified oligonucleotide comprises adifferent fluorophore. The formation of the biomolecule-oligonucleotideconjugates may form an additional fluorophore and/or chromophore duringthe conjugation reaction.

In certain embodiments, the method of preparing, purifying, and/orisolating a detectable component may be simple, high yielding orcombinations thereof. For example, the method may yield at least 30%40%, 50%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% isolated detectablecomponent. In other methods, the purity of the prepared, purified,and/or isolated detectable component may be at least 40%, 50%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%.

Detection

Suitable methods of detection may also include direct and/or indirectdetection of the oligonucleotide or oligonucleotides of the molecularprobe. For example by the use of DNA hybridization or DNA sequenceanalysis or DNA sequence amplification. In certain embodiments, theoligonucleotide or oligonucleotides may be detected by hybridization toan array of complementary oligonucleotides. In certain embodiments, theoligonucleotide or oligonucleotides may be detected by polymerization ofcomplementary DNA sequence. In certain embodiments, detection may be bythe use of immuno-PCR, a hybrid of PCR and immunoassay systems, whichcombines the versatile molecular recognition of antibodies with theamplification potential of DNA replication. The technique of immuno-PCRinvolves the in-situ assembly of the labeled DNA-antibody complex duringthe assay, creating variable stoichiometry in both the attachment of theDNA label, and the assembly of the components. For example, thepurified, labeled DNA may be added to a hybridization solutioncontaining denatured nucleic acids (RNA or DNA) from a sample to betested. The aqueous conditions of the hybridization solution may beadjusted to allow nucleic acid hybridization or reannealing, therebyallowing the labeled molecules to hybridize with unlabeled,complementary sequence counterparts. Duplex formation can be monitoredby digestion with single strand-specific nucleases (such as S1nuclease). Recovery and quantitation of the resistant, i.e.,double-stranded, reannealed material provides a measure of the nucleicacid sequence tested for. The amount of hybridization may be a functionof the initial concentration of DNA and the time allowed forreannealing. Therefore, increased initial DNA concentrations can lead tosubstantially reduced hybridization times. This technique may be anadditional means to monitor the presence of a target in a sample, suchas an antigen, of interest in a detection method, such as a Western blotassay.

A suitable method of detection may comprise a multiplex assay, utilizingone or more molecular probes, one or more detectable components, and oneor more universal adapters, wherein the one or more molecular probes maycomprise identical oligonucleotide sequences that are complementary to afirst oligonucleotide sequence segment of the one or more universaladapters. In certain embodiments, the method of detecting may comprise amultiplex assay, utilizing one or more molecular probes, one or moreuniversal adapters, and one or more detectable components, wherein theone or more molecular probes comprise one or more biomoleculesconjugated to identical oligonucleotide sequences, wherein the one ormore universal adapters comprise identical first oligonucleotidesequence segments complementary to the oligonucleotide sequences of theone or more molecular probes and a second, unique oligonucleotidesequence segment complementary to the oligonucleotide sequence of theone or more detectable components, and wherein the one or moredetectable components comprise unique oligonucleotide sequencescomplementary to the second, unique oligonucleotide sequence segment ofthe one or more universal adapters.

A suitable method of detection of one or more molecular targets in asample may provide using one or more detectable components comprisingone or more signal generating moieties. For example, the method ofdetecting one or more molecular targets in a sample may provide usingone or more molecular probes, one or more detectable components, one ormore universal adapters, and/or one or more spacer groups, orcombinations thereof. In certain embodiments, the method of detectingone or more molecular targets in a sample, comprising using one or moremolecular probes, one or more detectable components, one or moreuniversal adapters, and/or one or more spacer groups, may be provided inmultiple layers to increase the flexibility of a detection system, toenhance and/or increase the signal from the one or more moleculartargets, such as to enhance and/or increase the signal generated fromthe one or more molecular probe bound targets, to enhance and/orincrease the efficiency of the signal generated from the one or moremolecular probe bound targets, to enhance and/or increase the efficiencyof the molecular probe binding the one or more molecular targets. Themethod of detecting may be compatible with one or more detectionsystems, such as, for example, singleplex and multiplex assays, such asimmunoassays, protein detection assays, immunodetection, enzyme linkedimmuno-assays (ELISA), immunomagnetic cellular depletion, immunomagneticcell capture, flow cytometry, immunohistochemistry (IHC),immunocytochemistry (ICC), in situ hybridization (ISH), ELISpot, enzymeimmuno-assays (EIA), blotting methods (e.g. Western, Southern,Southwestern, and Northern), arrays, bead arrays, multiplex bead array,microarray, antibody array, cellular array, solution phase capture,chemiluminescence detection, infrared detection, labeling insideelectrophoresis systems or on surfaces or arrays, PCR amplification,elongation followed by PCR amplification, precipitation,immunoprecipitation, co-immunoprecipitation, chromatinimmunoprecipitation, pretargeting imaging, therapeutic agent, nucleicacid hybridization assays, microspcopy, imaging, high content screening(HCS), other assay or detection formats, for example, that are useful inresearch as well as in diagnosing diseases or conditions, orcombinations or derivatives thereof. In certain embodiments, the methodof detecting may be compatible with one or more different types ofmolecular targets, molecular probes, detectable components, universaladapters, and/or spacer groups. The method of detecting one or moremolecular targets in a sample may be provided by an increased and/orenhanced signal generated from the one or more molecular probe boundtargets, by, for example, increasing the number of detectable componentsutilized to detect each molecular target, and/or by amplification of thesignal by the instrumentation utilized. The method of detecting one ormore molecular targets in a sample, may be provided by an increasedand/or enhanced signal generated from the one or more molecular probebound targets, for example, molecular probes comprising antibodies, forexample, molecular targets comprising antigens, wherein the signalgenerated and detected may be amplified by the antibody-antigen complex.

In certain embodiments, a method of detection of one or more moleculartargets in a complex sample using one or more detectable components andone or more molecular probes, may comprise a multiplex assay, such as amultiplex immundection assay. The time of conducting the method ofdetection from the start of preparation of the hybrids to the end ofdetection may be about 0.5-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2,2-5, 2-4 or 2-3 hours.

In certain embodiments, a method of detection of one or more moleculartargets in a sample may utilize one or more detectable components andone or more molecular probes, comprising biomolecule-oligonucleotideconjugates, comprising: i) forming the molecular probes at greater than80% efficiency from at least one or more modified biomolecules and atleast one or more modified oligonucleotides; ii) forming the detectablecomponents at greater than 80% efficiency from a modifiedoligonucleotide and least one or more signal generating moieties,wherein the modified oligonucleotide is complementary to the modifiedoligonucleotide of the formed molecular probe; iii) providing the formedbiomolecular probes to the sample comprising the one or more moleculartargets; iv) contacting the one or more molecular targets in the samplewith the formed molecular probes; iv) providing the detectable componentto the sample comprising the contacted molecular probes; v) hybridizingthe complementary oligonucleotide of the detectable component with theoligonucleotide of the contacted molecular probe; vi) detecting the oneor more signal generating moieties of the hybridized molecular probescontacted to the one or more molecular targets.

A suitable method for detection of one or more target molecules maycomprise extending the oligonucleotide by PCR methods prior todetection. For example, the method of detection may comprise determiningthe amount of hybridization product or extension product, for example,determining a quantified amount of hybridization product or extensionproduct. The one or more molecular probes may comprise a nucleic acidbinding protein identical to, or substantially identical to, a lacrepressor protein or fragment thereof, which binds to a functional lacOsubsequence in a hybrid nucleic acid, hybridization product or extensionproduct. The nucleic acid binding agent may comprise one or more signalgenerating moities.

A suitable method for detection of one or more target molecules maycomprise detecting disease-specific molecules, identifying whether acertain cell type in a sample carries a disease-specific marker, and/ordetermining progression of a disease or condition. For example, diseasespecific markers may be molecules expressed by diseased cells, such ascancer cells, but not by non-diseased cells. A disease specific markermay be a molecule expressed by a pathogenic organism but not the hostorganism invades, and sometimes is a molecule expressed by a cellinvaded by a pathogenic organism and not by host cells not invaded bythe organism. A molecular probe may specifically bind to acancer-specific molecule, such as a marker specific for hepatocarcinomacells. A molecular probe may specifically bind to molecules expressedspecifically by liver, colon, uterus, and kidney cells. Certainembodiments may be useful for determining cell types and organs that arediseased, and are useful for determining the extent to which a diseasehas spread. A molecular probe may specifically bind to a moleculespecific for a progressive stage of a disease and may be included in thediagnostic, such as a molecular probe that specifically binds to amolecule specific for metastatic cells but not non-metastatic cells. Amolecular probe may specifically bind to a molecular marker specific toa cell type, diseased cell, or organism, and various markers specific toa cell type, diseased cell, or organism may be selected as a target forthese diagnostic applications. For examples, specific markers mayinclude, but are not limited to, EBNAI a viral nuclear antigen found inEBV infected B-cells; S100P, S100A4, prostate stem cell antigen,lipocalin 2, claudins 3 and 4, and trefoil factors 1 and 2 in pancreaticadenocarcinoma; CD antigens, microphthalmia transcription factor (MITF),and members of the Bcl-2 family in neoplastic mast cells; a cell surfacemarker, such as CDs, HLAs; or intracellular markers such as actins andtubulins as a healthy cell marker. In certain embodiments, hybridizationproducts or extension products may be detected using various methodsdescribed herein.

Suitable methods of detection of one or more molecular targets,including the utilization of kits and/or systems, may be useful fortherapeutic applications. In certain therapeutic embodiments, one ormore molecular probes, one or more detectable components, may beprovided to an in vitro or ex vivo sample from a patient or administeredto a patient in vivo. In certain therapeutic embodiments, the provided,or administered one or more molecular probes and one or more detectablecomponents, may further comprise a universal adapter. One or moleculartargets may be bound by the one or more molecular probes, and detectedupon hybridization with the one or more detectable components and/oruniversal adapters and one or more detectable components. The detectablecomponents may comprise one or more signal generating moieties. Thehybridized product may be extended by endogenous enzymes present in thesample or subject, and sometimes is extended by exogenous componentsdelivered to the sample or subject (e.g., a polymerase and/ornucleotides, such as a PCR method).

A suitable method for identifying a disease or condition in a subjectmay comprise delivering a first molecular probe and a second molecularprobe to a subject, wherein the first molecular probe comprises a firstbinding moiety partner and a first oligonucleotide and the secondmolecular probe comprises a second binding moiety partner and a secondoligonucleotide. The method may further comprise a universal adapter.The first binding moiety partner and second binding moiety partnerspecifically bind to a first binding region or second binding region ina target molecule, or a first target molecule and a second targetmolecule, where each target molecule may be independently selected froma target molecule specifically expressed by a diseased cell, a targetmolecule specifically expressed by a pathogenic organism, and a targetmolecule specifically expressed by a certain cell type. The firstoligonucleotide may comprise a first oligonucleotide sequencecomplementary to a second oligonucleotide sequence in the secondoligonucleotide and may be capable of forming a hybridized product withthe second oligonucleotide when the first hybrid is bound to the firsttarget molecule or first target molecular region and the second hybridis bound to the second target molecule or second target molecularregion, and the first target molecule and the second target molecule arein proximity or the first target molecular region and second targetmolecular region are in proximity. The hybridized product may beextended by delivering exogenous components that extend the hybridizedproduct (e.g., a polymerase and/or nucleotides, such as a PCR method),and a targeting component that may specifically bind to anoligonucleotide sequence in the hybridized product or extension productmay be delivered. The targeting component may comprise one or moresignal generating moieties, and the targeting component may be detectedby delivering a secondary agent, such as a secondary antibody, thatspecifically binds to the targeting component and comprises one or moresignal generating moieties. The hybrids, targeting component, and otherdiagnostic components may be delivered in an amount effective toidentify the disease or condition in the subject and/or patient.

A suitable kit for detection of one or more molecular targets in asample may comprise preparing, purifying, and/or isolating, one or moremolecular probes, one or more universal adapters, and/or one or moredetectable components, each of which may comprise one or more spacergroups, and providing to the sample the prepared, purified, and/orisolated one or more molecular probes, one or more universal adapters,and/or one or more detectable components, each of which may comprise oneor more spacer groups. For example, the kit for detecting one or moremolecular targets in a sample may be utilized in a method of detection.A suitable kit and/or system for detecting one or more molecular targetsin a sample, may comprise one or more prepared, purified and/or isolatedmolecular probes, such as one or more biomolecule-oligonucleotideconjugates, for example, antibody-oligonucleotide conjugates,protein-oligonucleotide conjugates, or peptide-oligonucleotideconjugates, one or more prepared, purified and/or isolated universaladapters, and/or one or more prepared, purified and/or isolateddetectable components, wherein each of the molecular probes, universaladapters, and/or detectable components may comprise one or more spacergroups. The kit and/or system for detecting one or more moleculartargets in a sample may be used in a method of detecting one or moremolecular targets in a sample. For example, the method of detecting theone or more molecular targets may comprise utilizing one or more of thefollowing detection techniques and/or methods, including, but is notlimited to: singleplex and multiplex assays, such as immunoassays,protein detection assays, immunodetection, enzyme linked immuno-assays(ELISA), immunomagnetic cellular depletion, immunomagnetic cell capture,flow cytometry, immunohistochemistry (IHC), immunocytochemistry (ICC),in situ hybridization (ISH), ELISpot, enzyme immuno-assays (EIA),blotting methods (e.g. Western, Southern, Southwestern, and Northern),arrays, bead arrays, multiplex bead array, microarray, antibody array,cellular array, solution phase capture, chemiluminescence detection,infrared detection, labeling inside electrophoresis systems or onsurfaces or arrays, PCR amplification, elongation followed by PCRamplification, precipitation, immunoprecipitation,co-immunoprecipitation, chromatin immunoprecipitation, pretargetingimaging, therapeutic agent, nucleic acid hybridization assays,microspcopy, imaging, high content screening (HCS), other assay ordetection formats, for example, that are useful in research as well asin diagnosing diseases or conditions, or combinations or derivativesthereof and/or combinations thereof. The kit for detecting one or moremolecular targets in a sample may further comprise one or more bindingmoieties. The kit for detecting one or more molecular targets in asample may further comprise one or more signal generating moieties. Thekit for detecting one or more molecular targets in a sample may furthercomprise one or more scaffolds, wherein the one or more scaffolds maycomprise one or more signal generating moieties.

In certain embodiments, a plurality of molecular probes, comprisingbinding moieties conjugated to oligonucleotides, and a plurality ofdetectable components, comprising signal generating moieties conjugatedto complementary oligonucleotides, may be preassembled, i.e., combinedand allowed to hybridize to form a composition comprising a plurality ofhybridized molecular probe-detectable components, that may then befollowed by contacting the preassembled composition with a sample,comprising one or more molecular targets. In certain embodiments, thepreassembled composition, comprising a plurality of hybridized molecularprobe-detectable components, may then be used in an assay to performboth recognition and detection functions. In certain embodiments, theplurality of molecular probes may comprise one or more antibodies. Incertain embodiments, the plurality of detectable components may compriseone or more signal generating moieties. The plurality of molecularprobes and/or the plurality of detectable components may comprise one ormore spacer groups. The plurality of molecular probes may comprise, forexample, 2 or more molecular probes, such as 3 or more, 4 or more, 5 ormore, 10 or more, 25 or more, 100 or more molecular probes. Similarly,the plurality of detectable components may comprise, for example, 2 ormore detectable components, such as 3 or more, 4 or more, 5 or more, 10or more, 25 or more, 100 or more detectable components.

In certain embodiments, the oligonucleotides of the plurality ofmolecular probes and the plurality of detectable components may not becomplementary, for example, the plurality of detectable components maycomprise signal generating moieties conjugated to oligonucleotides thatare not complementary to the oligonucleotides of the plurality ofmolecular probes. When the plurality of molecular probes and theplurality of detectable components comprise oligonucleotides that arenon-complementary, these may be combined together along with a pluralityof adaptor oligonucleotides. The plurality of adaptor oligonucleotidesmay comprise oligonucleotides sequence segments that may becomplementary to the plurality of molecular probes and oligonucleotidessequence segments that may be complementary to the plurality ofdetectable components. The plurality of molecular probes, the pluralityof detectable components, and the plurality of adaptor oligonucleotides,may then be allowed to preassemble, i.e., hybridize to form acomposition comprising a plurality of molecular probe-adapter-detectablecomponents, that may then be followed by contacting the composition witha sample, comprising one or more molecular targets.

In certain embodiments, a molecular probe may be combined with aplurality of detectable components, for example, 2 or more detectablecomponents, such as 3 or more, 4 or more, 5 or more, 10 or more, 25 ormore, 100 or more detectable components, to allow for detection in aplurality of assays, such as in a plurality of fluorescent channels, oralternatively, in a plurality of assay formats, such a fluorescent assayand an enzymatic activity assay. Similarly, a plurality of molecularprobes, for example, 2 or more molecular probes, such as 3 or more, 4 ormore, 5 or more, 10 or more, 25 or more, 100 or more molecular probes,may be combined with a detectable component, to allow for detection in afluorescent channel for samples containing a plurality of targets.

Preassembly Hybridization

In certain embodiments, the process of conducting the preassemblyhybridization prior to contacting a sample, may be advantageous forcertain applications, assays, and/or methods, and may lend itself tocreating novel formats, tests, assays, and/or classes of products.Preassembly may allow the formation of the molecular probe-detectablecomponents, and their purification and/or validation, in a differenttime or place from their actual use or application in an assay, whichmay be of significant value. The process of preassembly may beperformed, for example, by the end-user immediately prior to its use inan assay, or separately for example, by a commercial supplier which maythen be provided to the end user.

Advantageously, the process of preassembly may allow for the use of astrategy for multiplexed detection, wherein one or more molecular probesmay be combined with one or more detectable components on an as-neededbasis. For example, with a selection of one or more detectablecomponents available, such as 2 or more detectable components, such as 3or more, 4 or more, 5 or more, 10 or more, 25 or more, 100 or moredetectable components available, it may be possible to select a specificdetectable component to be associated with one or more molecular probes,such as in an assay comprised of a plurality of molecular probes to becontacted with a complex sample comprising one or more moleculartargets. In certain embodiments, a plurality of detectable componentsmay be prepared by conjugating a unique oligonucleotide to a pluralityof signal generating moieties, for example a plurality of scaffoldscomprising unique organic fluorophores, such that the plurality ofdetectable components correspond to a plurality of channels of acommercial flow cytometer. Then, to match a specific binding moiety,such as an antibody, to a particular channel, a molecular probe and thedetectable component bearing the appropriate fluorophore would bepreassembled. By conducting the process of preassembly, appropriatelymatching particular binding moieties to particular detectablecomponents, a panel of unique tests may be prepared in advance to allowfor a multiplexed assay, for example a multiplexed assay of a pattern ofimmunoreactivities on a population of cells, such as would be used inimmunophenotyping by flow cytometry. In addition, in certainembodiments, based on the results of a first multiplexed assay, a secondpanel of hybrids may be preassembled to further characterize the sample.In certain embodiments, one or more panels of preassembled hybrids maybe used to characterize a sample comprising one or more moleculartargets, such as 2 or more panels, for example, 3 or more, 4 or more, 5or more, 10 or more, 25 or more, 100 or more panels.

In certain embodiments, the process of preassembly may be readilyadaptable to automation. For example, a plurality of molecular probes,such as 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 25 ormore, 100 or more, 1,000 or more, or 10,000 or more molecular probes,may be placed into individual containers or wells that may then beaddressed individually by a robotic tool. Similarly, a plurality ofdetectable components, such as 2 or more, 3 or more, 4 or more, 5 ormore, 10 or more, 25 or more, 100 or more, 1,000 or more, or 10,000 ormore detectable components may be placed into individual containers orwells that may then be addressed individually by a robotic tool. Fromthese pluralities of individual molecular probes and individualdetectable components, a plurality of predetermined combinations, forexample predetermined by a table or a computation, may be preassembled.For example, a plurality of unique combinations or sets of molecularprobes and detectable components may be brought together by combining analiquot of a specific molecular probe and an aliqout of a specificdetectable component, in another container or well to allow theirhybridization to form a complex, wherein the amounts employed providethe appropriate ratios of oligonucleotides to allow properstoichiometry. In certain embodiments, the automated system may performthe steps of mixing to match the specific binding moiety, such as anantibody, to a particular fluorescence channel, to form a panel or setunder the control of a user. Alternatively, the automated system mightselect the specific binding moiety, such as an antibody, and match it toa particular fluorescence channel, to form a panel or set according toan algorithm. In certain embodiments, the automated system may performthe steps of mixing to match each specific binding moiety, such as anantibody, to a particular fluorescence channel, to form a panel or setunder the control of a user. Alternatively, the automated system mightselect each specific binding moiety, such as an antibody, and match itto a particular channel, to form a panel or set according to analgorithm. Using a preassembly format, a plurality of these sets, suchas two or more sets, three or more, four or more, five or more, ten ormore, twenty or more, fifty or more, one hundred or more, one thousandor more, or ten thousand or more sets may be preassembled on anas-needed basis to then be used, for example used in an assay.Advantageously, use of unique preassembled sets of molecular probes anddetectable components, automated multiplexed immunodetection assays,such as in flow cytometry, imaging, microscopy, high content screening(HCS), ELISA, ELISpot, or immunohistochemistry, to examine populationsand subpopulations of circulating lymphocytes, may be accomplishedrapidly, cost-effectively and/or with a minimal selection of reagents.Such an automated system may be able to perform a series of preassemblysteps, to create a panel of multiplexed sets that perform a defined oradaptive sequence of tests to characterize a sample. In certainembodiments, the automated system may perform, for example,immunophenotyping by subjecting the sample to analysis by multiple sets,formed in order by the process of preassembly, that are defined by adefined protocol. Alternatively, an automated system may select at leastin part the constituents of one or more sets to be used forimmunophenotyping based at least in part on the results of one or moreprevious sets, by applying an algorithm that incorporates at least inpart the one or more prior results to determine at least in part one ormore subsequent set, and then using the principles of preassembly, toform one or more additional sets as needed, thereby achieving speed andspecificity that exceed what can be obtained by other techniques.

Preparation, Purification and/or Isolation

In certain embodiments, the antibody-oligonucleotide conjugates may bepurified as depicted in FIG. 10. For example, the conjugation reactionmixture, comprising antibody-oligonucleotide conjugates and excessmodified oligonucleotide may be purified by binding theantibody-oligonucleotide conjugates to a column comprising agarose andmetal ions immobilized within the stationary phase of the column (whichmay be called “magnetic agarose” or “magnetic affinity beads”). Theprepared antibody-oligonucleotide conjugates may include moieties, suchas a histidine rich region, that may bind to metal ions that areimmobilized on the stationary phase of the column—which may now beseparated from the excess modified oligonucleotide, which do not havefunctionality that may bind to the metal ions in a similar chelatingfashion. Once the excess modified oligonucleotide has been washed by aseries of elutions, the bound antibody-oligonucleotide conjugates may bereleased by eluting with a displacing agent, such as another chelatingmoiety, for example, EDTA.

In certain embodiments, the modified oligonucleotides may be prepared asdepicted in FIG. 11. For example, in Stage 1, the modifiedoligonucleotides may be prepared by resuspending anamino-oligonucleotide in a buffer (Buffer A). The oligonucleotideconcentration (OD260/μL) may be determined by spectrophotometermeasurement. Once the concentration has been determined, the buffersolution may exchanged by sequential centrifuge spin down andresuspension of the resulting pellet in Buffer B to prepare for reactingwith the modifying reagent, followed by measuring the oligonucleotideconcentration (OD260/μL) in Buffer B by spectrophotometer measurement.Modification of the oligonucleotide may be conducted, for example, withS-4FB, using dimethylformamide (DMF) as a cosolvent. Once the reactionhas completed, the reaction mixture may be spun down and the Buffer Cexchanged into the system. Finally, the modified-oligonucleotide(4FB-modified oligonucleotide) concentration can be measured (OD260/μL)by spectrophotometer measurement, now in Buffer C.

In certain embodiments, the modified oligonucleotide may be prepared bysolid phase synthesis. The solid phase synthesis may also include thedirect incorporation of a linker during the solid phase oligonucleotidesynthesis. The solid phase synthesis may also include the directincorporation of a linker during the solid phase modifiedoligonucleotide synthesis.

In certain embodiments, the modified antibody may be prepared asdepicted in FIG. 12. For example, in Stage 2, the modified antibodiesmay be prepared by resuspending the antibody in a buffer (for example100 μg antibody at 1 mg/mL concentration). The antibody concentration(A280) may be determined by spectrophotometer measurement. Once theconcentration has been determined, the buffer solution may exchanged bysequential centrifuge spin down and resuspension of the resulting pelletin Buffer B to prepare for reacting with the modifying reagent, forexample, with S-HyNic. Once the reaction to modify the antibody has beencompleted, the reaction mixture may be spun down and the modifiedantibody, for example a S-HyNic-modified antibody, may be exchanged intoBuffer C. Finally, the modified-antibody concentration, for example, theS-HyNic-modified antibody concentration, may be measured by aspectrophotometer measurement, now in Buffer C.

In certain embodiments, the conjugation of a modified antibody with amodified oligonucleotide may be conducted as depicted in Stage 3 in FIG.13. For example, in Stage 3, the modified-antibody, a S-HyNic-modifiedantibody, may be reacted with an excess of the modified-oligonucleotide(4FB-modified oligonucleotide), to form antibody-oligonucleotideconjugates having at least one oligonucleotide conjugated to eachmodified-antibody. The reaction mixture will also have unreactedmodified-oligonucleotide (4FB-modified oligonucleotide).

In certain embodiments, the purification and isolation ofantibody-oligonucleotide conjugates may be conducted as depicted inStage 4 in FIG. 13. For example, in Stage 4, the conjugation reactionmixture, comprising antibody-oligonucleotide conjugates and excessunreacted modified-oligonucleotides (4FB-modified oligonucleotide), maybe placed in contact with “magnetic affinity beads,” for example, beadshaving metal ions immobilized that are available to be boundselectively, by chelation, with the product antibody-oligonucleotideconjugates but not with the unreacted modified-oligonucleotides. Oncethe antibody-oligonucleotide conjugates have been bound to the magneticaffinity beads, the beads are washed to remove the remaining reactioncomponents other than the bound antibody-oligonucleotide conjugates. Theantibody-oligonucleotide conjugates are then released with a displacingagent, such as Buffer D, which then is buffered exchanged with Buffer Evia sequential spin down and resuspension series, to provide purifiedantibody-oligonucleotide conjugates.

In certain embodiments, the protein-oligonucleotide conjugates may beprepared or purified, or both, as depicted in FIGS. 9, 10, 12 and 13,where a protein is modified rather than an antibody, and utilizingmodified oligonucleotides as depicted in FIGS. 9, 10, 11 and 13.

In certain embodiments, an antibody such as a monoclonal antibodydirected against a specific antigen of interest may be conjugated to anoligonucleotide to form a conjugate. In FIG. 14, the process for formingan antibody-oligonucleotide conjugate is presented diagrammatically.Here, the conjugation takes advantage of the chemical reaction betweenthe HyNic and 4FB moieties to promote full conversion of the antibody toconjugate. (A) represents a post-synthetic chemical modification methodto prepare a 4FB modified oligonucleotide. Here, anamino-oligonucleotide, for example, a C₆₋amino-oligonucleotide, thatencodes a specific barcode tag sequence is prepared by solid phasephosphoramidite chemistry. Then, using the N-hydroxysuccinimidereactivity of sulfo-succinimidyl activated 4-formyl benzoate (S-4FB),the oligonucleotide is modified and activated. Alternatively, in (B),the oligonucleotide is synthesized by solid phase phosphoramiditechemistry with a terminal 4FB phosphoramidite monomer. Theoligonucleotide may be of a number of different lengths to incorporateone or more barcodes, chemistries to incorporate alternative backbones,bases, or inert linkers, or geometries, such as tandem repeats orbranched dendrimers to allow incorporation of multiple copies of thebarcode sequence, as can readily be formed by standard means. Further,the 4FB moiety might be incorporated by a number of alternativechemistries or by biochemical means using enzymes. Further, a 4FB moietymight be placed at either the 3′ or 5′ end, or in the middle or close toeither end of an oligonucleotide. In parallel, in (C), the antibody orother protein, biomolecule, or other probe would be reacted toincorporate one or more HyNic moieties as via reaction of theN-hydroxysuccinimide reactivity of sulfo-succinimidyl activated6-hydrazinopyridine-3-carboxylate (S-HyNic) with a primary amine, suchas a Lysine amino acid epsilon amino group which are prevalent on thesurface of proteins. Five mole equivalents of S-HyNic to each moleequivalent of antibody might be used. Other chemical or biochemicalmeans could be used to modify the antibody or other probe molecule todisplay one or more HyNic moieties. Then, after purifying the4FB-modified oligonucleotide and HyNic-modified antibody, they can bebrought together, typically with a molar excess of the oligonucleotideto the antibody. Via the formation of the bisarylhydrazone bond, theantibody-oligonucleotide conjugate (D) is formed. As shown in (E),non-denaturing polyacrylamide gel electrophoresis of the HyNic modifiedantibody formed in (C) in Lane 2 reveals a single prominent band atapproximately 200 kD apparent mass. In Lane 3, theantibody-oligonucleotide conjugate formed in (D) demonstrates multiplebands indicating multiple molecular forms. Here the one mole equivalentof HyNic-antibody was combined with approximately three mole equivalentsof 4FB-oligonucleotide. As indicated by the numbering, the bands areconsistent with the conjugation of 0, 1, 2, or 3 oligonucleotides to theantibody. Thus, this process yields a mixture of HyNic-modifiedantibody, 4FB-oligonucleotide and antibody-oligonucleotide conjugateswith one or more oligonucleotides coupled to each antibody. By varyingthe mole ratio of S-HyNic to antibody and of 4FB-modifiedoligonucleotide to HyNic-modified antibody, essentially all, or nearlyall, of the antibody can be converted to oligonucleotide conjugate.

In certain embodiments, the purification and isolation ofantibody-oligonucleotide conjugates obtained by the reaction ofHyNic-modified antibody with a mole excess of 4FB-modifiedoligonucleotide may be desirable, as shown in FIG. 15. A chemicalseparation may be conducted in order to isolate theantibody-oligonucleotide conjugate away from unincorporatedoligonucleotide. For example the conjugation reaction mixture (A),comprising antibody-oligonucleotide conjugates and excess unreacted4FB-modified-oligonucleotides, may be placed in contact with “magneticaffinity beads,” for example, beads having metal ions immobilized bychelation that are available to be bound selectively with a binding siteon the antibody. Thus, the product antibody-oligonucleotide conjugateswill be substantially captured onto the beads and the unreacted 4FBmodified-oligonucleotides will not. Once the antibody-oligonucleotideconjugates have been bound to the magnetic affinity beads, the beads arewashed to remove the remaining reaction components other than the boundantibody-oligonucleotide conjugates. The antibody-oligonucleotideconjugates are then released with a displacing agent, such as Buffer D,which then is buffer-exchanged with storage Buffer E by applying thesolution to a centrifugal desalting column pre-equilibrated with BufferE. The eluent after centrifugation yields the purifiedantibody-oligonucleotide conjugate.

Immunodetection Assays and/or Detection

In certain embodiments, 1/1 antibody-oligonucleotide conjugates may berequired for immunodetection assays. Site specific 1/1antibody-oligonucleotides conjugates can be prepared as schematicallypresented in FIG. 16 wherein antibodies are reduced under controlledconditions to reduce two exposed disulfide bonds in the hinge region,followed by quenching of the reduced protein with MHPH, a thiol reactivearomatic hydrazine bifunctional modification reagent, followed bydesalting and conjugation to a 4FB-modified oligonucleotide in thepresence of aniline catalysis. In a non-site selective procedure, theantibody is controllably modified with S-HyNic to incorporate <3 HyNicmoieties followed by conjugation to 0.75 or less mole equivalents of a4FB-oligonucleotide in the presence or absence of aniline catalyst, theunconjugated oligonucleotide is removed by size exclusionchromatography, the unconjugated antibody is removed by ion exchangechromatography and the conjugate released from the cationic support toisolate the pure antibody-oligonucleotide conjugate.

In certain embodiments, protein binders other than full antibodiesincluding Fab′, Fab′-2 that possess free cysteine moities, proteinbinders such as scFvs, monobodies, nanobodies, diabodies and camelidsengineered to incorporate a single cysteine, or proteins engineered toincorporate unnatural amino acids such as acetyl-phenylalanineincorporated using engineered tRNAs, and aptamers can be barcoded witholigonucletotides and employed in immunodetection assays. FIG. 17presents schematically a procedure to incorporate a singleoligonucleotide barcode on a protein containing a single cysteine usingthe HyNic/4FB couple to produce an 1/1 protein-oligonucleotide conjugatemediated by an bis-arylhydrazone bond.

In certain embodiments, a variety of signal generators can be conjugatedto the complementary oligonucleotide. Oligonucleotide-signal generatorconjugates can also be prepared using the HyNic-4FB couple. FIG. 18presents schematically a method wherein an amino-substituted signalgenerator is modified to incorporate a HyNic moiety and is conjugated toa 4FB-oligonucleotide in the presence or absence of aniline catalyst.Other methods may also be employed. Signal generators that may beincorporated on complementary oligonucleotides, such as those of FIG.18, include but are not limited to, fluorescent protein; fluorophore;fluorosphere; quantum dot; enzyme; nucleic acid; scaffold; dendrimer;hydrogel; buckyballs; nanoparticles; nanogold; colloidal gold;microparticle; magnetic particle; bead; microarray; microfluidic device;wetted surface; biological cells; or derivatives or combinationsthereof.

In certain embodiments, complementary detectors can be prepared whereinthe detector construct is prepared such that the stoichiometry of theconstruct is 1 oligonucleotide conjugated to a single scaffold to whichmultiple signal generators are covalently bound. FIG. 19 presents thescheme described in the examples that was used. The complementarydetector was prepared in the following multi-step protocol: (A)amino-dextran, a 50,000 mean molecular weight polysaccharide bearing 40to 50 amine groups per molecule, was modified by reaction withsulfo-succinimidyl activated 6-hydrazinopyridine-3-carboxylate (S-HyNic)to incorporate 2-3 HyNic groups per molecule; (B) to each moleequivalent of HyNic-amino-dextran was added 0.5 mole equivalents ofcomplementary 4FB-modified oligonucleotide; (C) unconjugated4FB-oligonucleotide was removed by size exclusion column chromatography;(D) unconjugated dextran was removed by ion exchange columnchromatography in which the oligonucleotide-amino-dextran conjugate wasadsorbed on the cationic support, the unconjugated dextran was washedaway and the oligonucleotide-amino dextran conjugate was eluted from thesupport; (E) the oligonucleotide-amino dextran conjugate was exchangedinto pH 7.4 phosphate buffer and remaining amino groups on the dextrancomponent of the oligonucleotide-amino dextran conjugate were thenmodified by combining for each mole equivalent ofoligonucleotide-dextran conjugate, 5 mole equivalents of anN-hydroxysuccinimide-modified fluorescent organic dye; and the resultingoligonucleotide-dextran-fluorophore conjugate was purified by dialysis.

In certain embodiments, the antibody-oligonucleotide conjugates or otheroligonucleotide modified materials bearing a specific oligonucleotidesequence that serves as a specific barcode, may be combined withdetector conjugate composed of a complementary oligonucleotide sequencethat serves as the anti-barcode, chemically linked to a signalgenerator, such as an oligonucleotide-dextran-fluorophore conjugate.Given the principles of hybridization of complementary sequences, thisinteraction will result in DNA-directed self assembly, leading toformation of a complex where the antibody is stably associated with thesignal generator via a double stranded oligonucleotide linker. FIG. 20presents a schematic presentation of the process of mixing (A), anantibody-oligonucleotide conjugate formed by the reaction ofHyNic-modified antibody to a 4FB-modified oligonucleotide barcode, with(B), a complementary anti-barcode oligonucleotide similarly conjugatedto a signal generator. The interaction of the two oligonucleotides forms(C), a complex comprising an antibody now labeled with a signalgenerator, linked by the hybridized oligonucleotides. This interactionis documented by a native polyacrylamide gel electrophoresis analysis,(D). In Lanes 3 and 5, two distinct antibody-oligonucleotide conjugateseach demonstrate a range of species of characteristic mobility, e.g. A¹and A². As shown in Lanes 4 and 6, upon the addition of a complementaryoligonucleotide-dextran-fluorophore conjugate, the two antibodies appearin a new form with greatly decreased mobility, e.g. C¹ and C². Heregreater than 95% of each antibody-oligonucleotide conjugate hashybridized to the detector as indicated by the nearly quantitative shiftof the product to the slower mobility form.

Self-Assembly

In certain embodiments, the principle of self-assembly directed byhybridization between pairs of complementary oligonucleotides can beused to facilitate the independent formation of multiple complexes whereeach species represents a specific signal generator linked by a doublestranded oligonucleotide to a specific antibody. As diagrammed in FIG.21, in (A), multiple antibodies denoted Ab₁, Ab₂, Ab₃, Ab₄, etc., eachconjugated to a different barcode oligonucleotide denoted HyLk1, HyLk2,HyLk3, HyLk4, etc., might be applied as probes to interrogate a complexbiological sample. By the principle of binding of antibodies to theircognate antigen epitopes, the different antibody-oligonucleotideconjugates might interact with the sample to form distinct immunecomplexes that might distribute to distinct locations or be associatedwith distinct features, for example. Then, in (B), a set of anti-barcodeoligonucleotides comprising the complementary sequences, denoted asHyLk1′, HyLk2′, HyLk3′, HyLk4′, etc., and conjugated to different signalgenerators, denoted as SG₁, SG₂, SG₃, SG₄, etc., can be added. Then, in(C), by the principle of DNA directed self-assembly, each barcodeantibody would hybridize to its anti-barcode signal generator to formcomplexes. This would bring each signal generator into the distributionof each antibody, so that e.g. the distribution of Ab₁ could bedetermined by the distribution of SG₁, distribution of Ab₂ could bedetermined by the distribution of SG₂, etc.

In certain embodiments, the principle of self-assembly directed byhybridization between pairs of complementary oligonucleotides can beused to facilitate the independent formation of multiple complexes whereeach species represents a specific signal generator linked by a doublestranded oligonucleotide to a specific antibody. Thus, mixtures ofantibody-oligonucleotide conjugates can be used to detect one or moreantigens present on the surface of a living cell and then mixtures ofdetectors comprising the complementary oligonucleotide conjugated toreadily distinguished signal generators, in this case each a dextranscaffold modified with fluorophores of specific spectral properties, canbe applied to allow detection of the binding of each antibodyindependently in a single experiment, using the methodologies of flowcytometry. As diagrammed in FIG. 22, a sample of cells to becharacterized, such as mouse splenocytes, are treated with a mixture ofantibody-oligonucleotide probes, such as a mouse monoclonal antibodydirected against the mouse T helper cell surface glycoprotein CD4 (αCD4)conjugated to deoxyribose oligonucleotide HyLk1 and a mouse monoclonalantibody against the CD43 sialophorin characteristic of T cells (αCD43)conjugated to deoxyribose oligonucleotide HyLk2. After allowing time forbinding, the cells are washed so that the solution is free of unboundantibody-oligonucleotide conjugates. Then a mixture of complementaryoligonucleotide detectors which are conjugated to fluorescent proteinsor fluorescent dextrans, here a HyLk1′ conjugated to signal generator 1(SG¹) and HyLk2′ conjugated to signal generator 2 (SG²), are added todetect the bound antibodies. The resulting hybridization of HyLk1 toHyLk1′ and HyLk2 to HyLk2′ then links SG¹ to cells presenting CD4 andSG² to cells presenting CD43. As shown here, the cell indicated would beidentified by flow cytometry as displaying fluorescence from both signalgenerators SG¹ and SG², which would then lead to the conclusion thatthis cells is potentially a CD4⁺ CD43⁺ T cell.

Detectors Comprising the Complementary Oligonucleotide

In certain embodiments, antibody-oligonucleotide conjugates anddetectors comprising the complementary oligonucleotide conjugated to asignal generator, in this case a complementary oligonucleotide HyLk1′conjugated by bisarylhydrazone linkage to R-phycoerythrin (R-PE), abiofluorescent protein, can be used to detect the CD4 cell surfaceprotein on splenocytes in flow cytometry. FIG. 23 compares a set of twodimensional flow cytometry plots, where each cell that is detected isindicated by a dot representing its specific forward scatter. FSCdenoted on the Y-axis, and its specific fluorescence in the R-PEchannel, denoted by R-PE on the X axis. The dots represent approximately10,000 individual cells examined in a single experiment. As shown inplot (A) in the upper left, when splenocytes are gated to select forlymphocytes, they demonstrate a characteristic forward scatter ofapproximately 300 FSC Units, and display approximately a fluorescenceintensity in the R-PE fluorescence channel of 5 R-PE Units. Here, thevalue of 5 R-PE Units represents the background due to endogenousfluorescence, limitations of the instrumentation and other features. Inthe flow cytometry plot (B) on the upper right, addition of 30 ng ofHyLk1′ conjugated to R-PE detector to the splenocytes causes thelymphocytes to display increased fluorescence in the R-PE channel, witha median intensity of approximately 10 R-PE Units. This controlexperiment reveals the detector background signal, which may be ascribedto non-specific binding of the detectors to the lymphocytes. For theexperiment, in plot (C) in the lower panel, 0.1 μg of αCD4 conjugated bybisarylhydrazone chemistry to HyLk1 was first added to the splenocytesand allowed to bind. Then the cells were washed and treated with 30 ngof HyLk1′-R-PE. The cells were then analyzed by flow cytometry asbefore. Note that two populations of cells are detected, one with amedian intensity of 20 R-PE Units and a second with a median intensityof 3000 R-PE Units. The 3000 R-PE Unit population represents the subsetof splenocytes that would be considered as CD4 positive (CD4⁺) by thisassay, which normally identifies presumptive T helper cells. The 20 R-PEUnit population represents the cells that bound low levels of theantibody and/or the detector and are considered CD4 negative (CD4⁻), andrepresents the non-specific background in the experiment. A proxy forthe signal to background (S/B) of this experiment can be estimated asthe ratio of the median intensity of the CD4⁺ and CD4⁻ populations, CD4⁺_(i)/CD4⁻ _(i), which here would be calculated as greater than (>) 100.It is common to consider that a S/B>100 is a characteristic of a highquality biochemical assay.

In certain embodiments, antibody-oligonucleotide conjugates anddetectors comprising the complementary oligonucleotide conjugated to asignal generator, in this case a complementary oligonucleotideconjugated to the biofluorescent protein allophycocyanin (APC), can beused to detect biomarker in flow cytometry. As shown in FIG. 24, theplot (A) in the upper left indicates the background fluorescence forlymphocytes, which has a median value of approximately 5 APC Units. Theplot (B) in the upper right indicates that after addition of 10 ng ofHyLk1′ conjugated by bisarylhydrazone chemistry to APC (HyLk1′-APC), themedian fluorescence does not increase appreciably and remains at 5 APCUnits, indicating that the non-specific binding of the detector isnegligible. The lower plot (C) indicates the results when splenocyteswere treated with 2 μg of αCD4 conjugated to HyLk1, washed, treated with10 ng of HyLk1′-APC and analyzed by flow cytometry. Here, the twopopulations indicate a CD4⁻ population of cells at approximately 10 APCUnits and a CD4⁺ population at 100 APC Units. The S/B here is estimatedat approximately 20.

In certain embodiments, antibody-oligonucleotide conjugates anddetectors comprising the complementary oligonucleotide conjugated to asignal generator, in this case a complementary oligonucleotideconjugated to a dextran scaffold to which multiple DyLite 490fluorophores have been coupled, can be used to detect biomarker in flowcytometry. As shown in FIG. 25, the plot (A) in the upper left indicatesthe background fluorescence for lymphocytes, which has a median value ofapproximately 5 Dy490 Units. The plot (B) in the upper right indicatesthat after addition of 10 ng of HyLk1′ conjugated by bisarylhydrazonechemistry to dextran which was then labeled with DyLite 490(HyLk1′-poly-Dy490), the median fluorescence increases to 10 Dy490Units, indicating measurable non-specific binding of the detector. Thelower plot (C) indicates the results when splenocytes were treated with2 μg of αCD4 conjugated to HyLk1, washed, treated with 10 ng ofHyLk1′-poly-Dy490 and analyzed by flow cytometry. Here, the twopopulations indicate a CD4⁻ population of cells at approximately 10Dy490 Units and a CD4⁺ population at 500 Dy490 Units. The S/B here isestimated at approximately 50.

Flow Cytometry, Western Blot, Library of Monoclonal Antibodies, Beads,ELISA

A limiting feature for success of flow cytometry analysis to detectantigens present in or on individual cells is the sensitivity andspecificity of detection of that antigen. In general, antibodies arecommonly used as probes, given their properties as sensitive andspecific detection reagents. When the antibodies are renderedfluorescent, they may be detected by flow cytometry. Direct fluorescentlabeling of antibodies to form stable, covalent antibody-fluorophoreconjugates such as FITC conjugates, R-PE conjugates, APC conjugates orothers allows their facile use in flow cytometry, but may alter thefavorable properties of the antibody as a detection reagent. Inparticular, conjugation to multiple small organic fluorophores mayinactivate a significant fraction of antibodies or alter solubility.Conjugation to fluorescent proteins R-PE or APC may impair accessibilityof the antibody combining site to antigen epitopes. Anantibody-oligonucleotide conjugate may be used for flow cytometry as analternative to an antibody-fluorophore conjugate. The detectors maycomprise the complementary oligonucleotide conjugated to a signalgenerator. These may comprise a scaffold molecule conjugated to multiplesmall organic fluorophores, such as HyLk1′ conjugated bybisarylhydrazone chemistry to dextran which was then labeled with DyLite490 (HyLk1′-poly-Dy490). As shown in FIG. 26, a comparison ofcommercially available antibody-fluorophore conjugates toantibody-oligonucleotide conjugates recognizing surface antigens CD4, aT helper cell surface antigen, and CD8, a cytotoxic T cell surfaceantigen, was performed. Alternatively, αCD4:FITC or αCD8:FITC wereapplied under standard conditions to splenocytes and the samplesubjected to flow cytometry. Then, a αCD4-HyLk1 conjugate or aαCD8-HyLk1 conjugate were applied to the splenocytes and the samplesubjected to flow cytometry. The graphs represent histograms summarizingthe flow cytometry data, gated to detect lymphocytes, obtained from (A),unstained splenocytes or stained using the αCD4:FITC or stained usingthe αCD4-HyLk1/HyLk1′-poly-Dy490 couple, and (B), unstained splenocytesor stained using αCD8:FITC or stained using theαCD8-HyLk1/HyLk1′-poly-Dy490 couple. The Y-axis represents the relativeabundance of cells that displayed a specific intensity of fluorescencein the FITC channel, as distributed on the X-axis. As shown in A and B,the αCD4:FITC and αCD8:FITC reagents displayed the favorable property ofa small fluorescence background with respect to cells that would beconsidered CD4⁺ or CD8⁺, respectively. By comparison, as shown in A andB, applying either the αCD4-HyLk1/HyLk1′-poly-Dy490 couple or theαCD8-HyLk1/HyLk1′-poly-Dy490 couple caused a shift of the cells toincreased signal in the FITC channel, including those that would beconsidered CD4⁺ or CD8⁺, respectively. This would be interpreted asnon-specific background, a potentially unfavorable feature. As shown inA, a similar fraction of cells in each population demonstrated a highstaining level when treated with αCD4:FITC orαCD4-HyLk1/HyLk1′-poly-Dy490, representing CD4⁺ cells. Similarly, asshown in B, a similar fraction of cells in each population demonstrateda high staining level when treated with αCD8:FITC orαCD8-HyLk1/HyLk1′-poly-Dy490, representing CD8⁺ cells. The medianintensity of CD4⁺ cells as detected by αCD4:FITC is significantly lessthan those detected by αCD4-HyLk1/HyLk1′-poly-Dy490. Similarly, themedian intensity of CD8⁺ cells as detected by αCD8:FITC is significantlyless than those detected by αCD8-HyLk1/HyLk1′-poly-Dy490. The higherintensity of cells that display positive staining would be interpretedas signal, a favorable feature. As a measure of sensitivity andspecificity and the quality of the assay, examining the ratio of medianintensity of the CD4⁺ to CD4⁻ cells or the CD8⁺ to CD8⁻ cells offers ameasurement of signal to background (S/B). Here, the commercial reagentsdisplay a S/B of ˜10 for αCD4:FITC and ˜50 for αCD8:FITC. Here, theoligonucleotide conjugates display a S/B of ˜50 forαCD4-HyLk1/HyLk1′-poly-Dy490 and ˜100 for αCD8-HyLk1/HyLk1′-poly-Dy490.These data suggest that the prototype olignucleotide conjugates andcomplementary oligonucleotide detectors compare well to existingcommercialized reagents as detection reagents.

In certain embodiments, more than one antibody-oligonucleotideconjugates will be brought into contact with detectors comprising onecomplementary oligonucleotide conjugated to a signal generator as wellas one or more non-complementary oligonucleotides conjugated to signalgenerators, as an alternative to multiple conventionalantibody-fluorophore conjugates used together to analyze multipleantigens in a single experiment. An advantage of the direct conjugationof the fluorescence signal generator to the antibody is the highpotential for correct identification of an antibody based on afluorescence signal alone. Under conditions where multipleantibody-oligonucleotide conjugates are used along with multipleoligonucleotide-signal generator conjugates, it may be desirable to haveno appreciable interaction between non-complementary pairs. As such, aconsideration in evaluating the antibody-oligonucleotide conjugates andoligonucleotide-signal generators is to investigate the potential forinteractions between pairs of non-complementary oligonucleotides leadingto false positive signals, commonly described as crosstalk. Towardtesting crosstalk between noncomplementary pairs, splenocytes werestained and analyzed by flow cytometry to compare the fluorescence oflymphocytes that were treated with no antibody, or with αCD8-HyLk2, andthen treated with the non-complementary probe HyLk1′-poly-Dy490 orHyLk1′-R-PE. In FIG. 27, the results of flow cytometry are displayed asplots of the relative incidence of cells on the Y-axis that display afluorescence intensity as indicated on the X-axis for the indicatedfluorescence channel, FITC or R-PE. In graph (A), the presence orabsence of αCD8-HyLk2 has no appreciable effect on the medianfluorescence after treatment with HyLk1′-poly-Dy490. Similarly, in (B),the presence or absence of αCD8-HyLk2 has no appreciable effect on themedian fluorescence after treatment with HyLk1′-R-PE. These resultssuggest that cross-talk is not a significant feature of non-specificbackground in experiments using antibody-oligonucleotide conjugates.

In certain embodiments, antibody-oligonucleotide conjugates anddetectors comprising the complementary oligonucleotide conjugated to asignal generator, in this case a scaffold modified with multiplefluorophores, can be used to detect cell surface antigens by flowcytometry as an alternative to conventional antibody-fluorophoreconjugates. Another perceived advantage of direct conjugation ofantibodies to fluorophores is that the binding of antibody to theantigen simultaneously, or substantially simultaneously, achieves thefluorescent labeling step, potentially saving time. Toward examining thespeed of interaction of antibody-oligonucleotide conjugates andcomplementary oligonucleotides conjugated to dextran scaffolds modifiedby fluorophores, an experiment was conducted where splenocytes stainedwith αCD8-HyLk2 were washed and then contacted with HyLk2′-poly-Dy549for specific times and then immediately introduced into the flowcytometer. The graphs (A) through (E) in FIG. 28 representtwo-dimensional flow cytometry plots of the experiment where (A)demonstrates the background signal prior to addition of theHyLk2′-poly-Dy549 detector and then (B), (C), (D) and (E) represent thestaining and detection of CD8⁺ cells at 1 minute, 5 minutes, 10 minutesand 15 minutes after addition of the HyLk2′-poly-Dy549 detector. Thedata from (A) to (E) are superimposed in (F) plotted as a histogram ofrelative abundance of cells at each fluorescence intensity, allowingdirect comparison. As can be seen, the addition of HyLk2′-poly-Dy549causes a shift of the CD8⁻ cells within one minute from a median valueof ˜3 Units to ˜7 Units. These cells do not become appreciably morefluorescent over the subsequent incubation. At 1 minute, the CD8⁺ cellsform a distinct population, indicated by the arrow in (B), that displaysa median intensity of ˜100 Units. At 5 minutes in (C), the CD8⁺population displays increased fluorescence to ˜200 Units. At 10 minutesin (D), the CD8⁺ population displays increased fluorescence to ˜300Units and at 15 minutes in (E), the fluorescence is ˜400 Units. Theseresults indicate, in certain embodiments, that incubation times as shortas 1 to 15 minutes are sufficient for hybridization of theoligonucleotides conjugated to dextran scaffolds modified byfluorophores to the antibody-oligonucleotide conjugates to permitdetection of antigens with high signal to background.

Depending on the particular application incubation times to permitsufficent detection may vary. In certain embodiments, incubation timesto permit sufficient detection may include overnight, 1 minute to 1hour, 5 minutes to 20 minutes, 30 minutes to 1 hour, 20 minutes to 2hours, 1 to 4 hours, 3 to 8 hours, or 6 to 12 hours.

In certain embodiments, antibody-oligonucleotide conjugates anddetectors comprising the complementary oligonucleotide conjugated to asignal generator, in this case a scaffold modified with multiplefluorophores, can be used to detect cell surface markers in flowcytometry to further characterize a cellular sample by exploitingalternative fluorophores that can be detected independently from anotherreference fluorophore. Here, each pair of antibody-oligonucleotideconjugates and complementary oligonucleotide conjugated to a signalgenerator may need to be examined to obtain the optimal signal tobackground, in a process of optimization. As shown in FIG. 29 ananti-CD19 antibody recognizing the CD 19 B lymphocyte surface antigen,conjugated to HyLk3 was added to splenocytes, allowed to bind, washedand subsequently the complementary HyLk3′ oligonucleotide coupled to adextran scaffold modified with Dy591 was added and the cells analyzed byflow cytometry. Shown are a set of four two-dimensional flow cytometryplots, where each cell that is detected is indicated by a dotrepresenting its specific side scatter, SSC denoted on the Y-axis, andits specific fluorescence in the channel detecting Dy591 on the X-axis.In A), the splenocytes were left unstained and gated for lymphocytes,demonstrating a single distribution of cells that display a medianintensity of ˜3 Dy591 intensity units. In B), C) and D), the splenocyteswere treated with 0.1 μg of αCD19-HyLk3 before washing and treating with0.3 μg, 0.1 μg or 0.03 μg of HyLk3′-poly-Dy591, respectively. Twosubpopulations stained cells can be distinguished in B) and C), thatrepresent CD19⁺ and CD19⁻ cells. In B), the CD19⁺ cells display a medianintensity of 100 units, while the CD 19⁻ cells are shifted due tonon-specific background to a median intensity of ˜20 units, yielding aS/B or ˜5. Here, the detector reagent would be considered to be presentin excess to an optimal amount. In C) the CD19⁺ cells display a medianintensity of ˜50 units, while the CD19⁻ cells are not appreciablyshifted due to non-specific background, yielding a S/B or ˜10. Here, thedetector would be considered to be close to an optimal amount. In D),the presumptive CD 19⁺ cells cannot be reliably distinguished from theCD19⁻ cells. Here, the detector would be considered to be below theoptimal amount.

In certain embodiments, antibody-oligonucleotide conjugates anddetectors comprising the complementary oligonucleotide conjugated to asignal generator, in this case a scaffold modified with multiplefluorophores, can be used to detect detect cell surface markers in flowcytometry to characterize a cellular sample. As shown in FIG. 30, asplenocyte preparation was examined to independently determine theproportion of cells that might be CD 19⁺ or CD8⁺, based on binding ofantibody-oligonucleotide conjugates. In plots A and B, the optimalconditions determined above of 0.1 μg of αCD19-HyLk3 and then 0.1 μg ofHyLk3′-poly-Dy591 were applied, yielding plot B. Note that the medianfluorescence intensity of the CD19⁻ fraction is only slightly greaterthan the unstained control in plot A. In plots C and D, 0.1 μg of ananti-CD8 antibody conjugated to HyLk2, αCD8-HyLk2, was added tosplenocytes, allowed to bind, washed and subsequently 0.1 μg of thecomplementary HyLk2′ oligonucleotide conjugated to the dextran scaffoldand modified with Dy549, HyLk2′-poly-Dy549, was added and the cellsanalyzed by flow cytometry, yielding plot D. Note that the medianfluorescence intensity of the CD19⁻ fraction is only slightly greaterthan the unstained control in plot C.

It is common in flow cytometry of complex cellular samples to detect twoor more surface antigens in a multiplex experiment, as a method todistinguish between cells with overlapping patterns of surface antigenexpression. FIG. 31 demonstrates an experiment wherein 5 commerciallysourced antibody-fluorophore are used to perform a multiplex flowcytometry experiment on mouse splenocytes. The antibodies recognize CD4(T cell receptor co-receptor MHC class II restricted, HIV receptor, Thelper cell antigen), CD8 (T cell receptor co-receptor, cytotoxic T cellantigen), CD19 (B lymphocyte surface antigen), CD43 (sialophorin,characteristic of both T and B lymphocytes), and CD62L (L-selectin,characteristic of T and B lymphocytes). In each of the two-dimensionalflow cytometry plots shown, cells gated to display only lymphocytes arerepresented by dots and their position with respect to the X-axisrepresents the intensity of the staining of each cell by theαCD4-APC.Cy7 tandem conjugate. In plot (A), the Y-axis displays thestaining with αCD8-PE. As shown, many lymphocytes are unstained byeither probe (lower left quadrant) and score as CD4⁻ CD8⁻, likelyrepresenting primarily B lymphocytes. Some lymphocytes can be identifiedas CD4⁺ (lower right quadrant) and some CD8⁺ (upper left quadrant) butfew, if any, are identified as CD4⁺ CD8⁺ (upper right quadrant). Thesesurface antigens are often restricted to different classes of T cells.In turn, a similar analysis applies to (B), where the Y-axis representsstaining with αCD19-APC. These results show an apparent lack of CD4⁺ CD19⁺ cells, recognizing that CD4 is a T cell antigen and CD19 is a B cellantigen. The cells in the lower left quadrant are likely to representprimarily CD8⁺ T cells. In (C), where the Y-axis represents stainingwith αCD43-FITC, some cells are scored as CD4⁺ CD43⁺, insofar as CD43 isa characteristic antigen of T cells, including CD4⁺ T cells. In (D),where the Y-axis represents staining with αCD62L-AF700, most CD4⁺ cellsare also CD62L⁺, as are most of the CD4⁻ cells including the CD8⁺ Tcells and the B cells.

In certain embodiments, multiple antibody-oligonucleotide conjugates andtheir complementary detectors can be used simultaneously, orsubstantially simultaneously, to detect two or more protein biomarkersin a multiplex experiment, as a method to distinguish between cells withoverlapping patterns of surface antigen expression. Here, the potentialof antibody-oligonucleotide conjugates and complementary oligonucleotidedetectors in such an assay is evaluated. As shown in FIG. 32,αCD4-HyLk1, αCD8-HyLk2, αCD19-HyLk3, αCD43-HyLk4 and αCD62L-HyLk5conjugates were added simultaneously to lymphocytes, allowed to bind andwashed. Subsequently, the five complementary oligo-dextran-fluorophoreconjugates, HyLk1′-poly-Dy490, HyLk2′-poly-Dy549, HyLk3′-poly-Dy591,HyLk4′-poly-Dy649 and HyLk5′-poly-Dy405 were added simultaneously,allowed to hybridize and the fluorescent signals were detected by flowcytometry. Here, the flow cytometry data are represented by a series oftwo dimensional plots, where each cell is represented by a dot at theposition of its intensity in the Dy490 (FITC) fluorescence channel asindicated on the X-axis and by intensity of the indicated fluorescencechannel on the Y-axis. In (A), the Y-axis represents αCD8 binding byDy549 fluorescence intensity. Both CD4⁺ and CD8⁺ cells are detected, butnot CD4⁺ CD8⁺ cells. In (B), the Y-axis represents αCD19 binding byDy591 fluorescence intensity, demonstrating the capability todistinguish CD 19⁺ B cells from CD4⁺ T cells. In (C), αCD43 binding isrepresented by Dy649 fluorescence on the Y-axis, demonstrating theresult that some CD43⁺ cells are CD4⁺ and that some CD4⁺ cells areCD43⁺. Finally, in (D), the binding of αCD62L is represented by Dy405fluorescence on the Y-axis, demonstrating the result that most CD4⁺cells are also CD62L⁺. Comparison of the distributions of the ability todistinguish markers and relative numbers of cells between the resultsusing commercial conjugates shown in FIG. 31 to using DNA directedassembly to form complexes between antibody-oligonucleotide conjugatesand complementary oligonucleotide detectors shown in FIG. 32 revealssimilar results.

In certain embodiments, antibody-oligonucleotide conjugates can be usedto bind to a cell surface antigen on a specific cell or type of cellthat may or may not be present in a mixture of cells and otherbiological components such as blood or another in a biological fluid,and these cells can be captured by hybridization to the complementaryoligonucleotide immobilized on a magnetic bead or other solid surface.Then, using a magnetic field, these cells can be removed from the restof the sample such as other cells, plasma, or other sample components.In some embodiments, the sample such as blood, depleted of the capturedcells, can then be used for a purpose such as transfusion. Here, themethod would be used for negative selection. In other embodiments, theisolated cells can then be used for some purpose such astransplantation, culture or subjected to analysis. Here, the methodwould be used for positive selection or cell enrichment. FIG. 33illustrates this concept. Here an α-CD4-HyLk1 conjugate is added to thesample and the conjugates selectively binds to the CD4⁺ T helper cellshown but not to other lymphocytes or other blood cells such asmonocytes, granulocytes, platelets or other blood cells. SubsequentlyHyLk1′-magnetic beads are added to the sample and the CD4⁺ cells boundto the magnetic bead are isolated away from the other cells byapplication of a magnet.

In certain embodiments, the sample, depleted of the captured cells, maythen be used for a purpose such as transfusion. Here, the method wouldbe used for negative selection. In other embodiments, the method wouldbe used for positive selection or cell enrichment, with the enrichedpopulation being used for downstream analysis. FIG. 80 illustrates thedepletion/enrichment concept, in which (A) Her2 antibody (Herceptin®,Genentech, California) conjugated to oligo HyLk1 is added to a complexblood sample. The Herceptin-HyLk1 conjugate selectively binds to Her2⁺tumor cells, but not to Her2⁻ blood cells, including leukocytes,monocytes, granulocytes, and platelets. HyLk1′-magnetic particles areadded to the sample (B), and HyLk1-antibody labeled Her2⁺ cells bound byhybridization to the magnetic particles are isolated from Her2⁻ cells byapplication of a magnet (C), resulting in a Her2-depleted sample and aHer2−enriched sample. In FIG. 82, depletion of human Her2+ tumor cellsfrom a sample of cultured leukocytes is illustrated using certainmethods described herein. 10% Her2− overexpressing human breastadenocarcinoma cells were spiked into a culture of human leukocytecells. The sample was (A) undepleted, (B, C) subjected to ‘mock’depletion methods without one or more necessary components, (D)Her2−depleted using conventional methods, (E) Her2− depleted usingconventional methods, modified with the 2-step approach included in theHybriLink strategy disclosed herein but without the use of oligos; or(F) Her2−depleted using HybriLink strategy disclosed herein. Followingtreatment, the samples were stained with fluorescent antibodies againsttumor cell markers Her2 (PE conjugate) and EpCAM (APC conjugate) todistinguish leukocytes (Her2−EpCAM−) from tumor cells (Her2+ EpCAM+).EpCAM was used as a secondary tumor cell marker to confirm depletion oftumor cells, given that binding of Herceptin during cellular labelingprior to magnetic depletion potentially interferes with downstream Her2staining, should the Her2 antibodies target the same protein epitope,thereby giving a false positive indication of successful depletion.Double-staining with αEpCAM provides a control against false depletionstaining. Following αHer2:PE/αEpCAM:APC staining, cells were analyzedusing a BD LSRII cytometer equipped with 561 nm and 633 nm lasers andappropriate optical fluorochrome filters. Raw data files were visualizedusing FlowJo software (TreeStar, Inc., Ashland, Oregon). Positivestaining gates were established based on fluorescent intensity of cellsstained with host IgG isotype-fluorochrome controls (Data not shown). Inundepleted cell population (A), 9.95% of cells are Her2+EpCAM+ tumorcells, and 68.5% are Her2− EpCAM−, for a tumor cell:leukocyte ratio of1:6.9. Samples subjected to “mock” depletion, in either the absence ofcomplementary oligo HyLk1′ on magnetic beads (panel B) or in the absenceof both oligo:IgG and complementary HyLk1′ oligo on beads (panel C) didnot exhibit successful depletion, indicating that beads unlabeled bycomplementary oligo have no nonspecific affinity for tumor cells, HyLk1oligonucleotide, or Herceptin antibody conjugates, any of which wouldinterfere with successful depletion. Samples depleted using currentstate-of-the-art methods (panel C), in which biotinylated Herceptin wasimmobilized on to streptavidin-surfaced magnetic nanospheres and appliedto cells, showed a 56% depletion of tumor cells, a 1:18.3 tumorcell/leukocyte ratio, and a depletion ratio of 2.7×. A modifiedstate-of-the-art method, in which cells were labeled byHerceptin-biotin, and then isolated using streptavidin surfacednanospheres, was less successful than conventional methods, resulting in32.2% (1.8×) Her2 depletion. However, samples depleted using theHybriLink depletion method described herein exhibited a tumor celldepletion of 77.9%, with a tumor cell/leukocyte ratio of 1:39.0, or 5.7×depletion ratio. The remaining tumor cell population was just 2.2%.Panel G summarizes statistical data contained herein. Example 31describes experimental methodology.

In certain embodiments, it may be necessary to both capture a specificcell type by immunomagnetic protocols and subsequently release the cellfor further analyses. Cells isolated by hybridization usingoligonucleotides prepared from natural nucleic acids as presented inFIG. 34, can be released from the magnetic beads by strand displacementof the hybrid formed between the antibody-oligonucleotide conjugatebound to the cell surface and the complementary oligonucleotide on thebead. Here, oligonucleotides based on peptide nucleic acid (PNAs),locked nucleic acid (LNAs), morpholino or other oligonucleotide analogsthat are capable of strand invasion of DNA duplexes will be designed tohybridize to the strand coupled to the magnetic bead releasing the cellfrom the bead. The isolated cells would subsequently be available fortransplantation, culture, or analysis by flow cytometry, imaging,microscopy, high content screening (HCS), ELISA, ELISpot, orimmunohistochemistry, or other assays. In certain embodiments, one ormultiple antibody-oligonucleotide conjugates and their complementarydetector-signal generator conjugates can be used to capture cells in amicrofluidic device in which complementary oligonucleotides areimmobilized in specific spots or on specific posts. Here, as presenteddiagrammatically in FIG. 35, using the example of the capture ofcirculating EpCam⁺ cancer cells, (A) anti-EpCam antibody-HyLk1 conjugateis added to a blood sample, allowed to bind and (B) the sample isallowed to flow through a microfluidic channel containing posts to whichHyLk1′ is immobilized and the anti-EpCam antibody-HyLk1 conjugate/EpCam⁺cell complex are captured by hybridization. Other cells, such aslymphocytes may flow through the microfluidic device.

In certain embodiments, cells may be released from the magneticparticles by strand displacement of the hybrid formed between theantibody-oligonucleotide conjugate bound to the cell surface and thecomplementary oligonucleotide on the bead. Here, oligonucleotides basedon peptide nucleic acid (PNAs), locked nucleic acid (LNAs), morpholinoor other oligonucleotide analogs that are capable of strand invasion ofDNA duplexes may designed to hybridize to the strand coupled to themagnetic bead releasing the cell from the bead. The isolated cells wouldsubsequently be available for transplantation, culture, or analysis byflow cytometry, imaging, microscopy, high content screening (HCS),ELISA, ELISpot, immunohistochemistry (IHC), or other assays. In certainembodiments, one or multiple antibody-oligonucleotide conjugates andtheir complementary detector-signal generator conjugates may be used tocapture cells in a microfluidic device in which complementaryoligonucleotides are immobilized in specific spots or on specific posts.Here, as presented diagrammatically in FIG. 83, using the example of thecapture of Her2⁺ cancer cells circulating in a complex whole bloodsample, (A) Her2 antibody-HyLk1 conjugate is added to the sample,allowed to bind, and (B) the sample is allowed to flow through amicrofluidic channel containing posts to which HyLk1′ is immobilized;thereby, HyLk1-Her2 antibody-Her2⁺ cell complexes are captured byhybridization, while Her2⁻ blood cells, such as leukocytes, monocytes,granulocytes, and platelets, may flow through the microfluidic device.

In certain embodiments, one or multiple antibody-oligonucleotideconjugates and their complementary detector-signal generator conjugatescan be used individually or simultaneously to detect one or more proteinbiomarkers in a single or multiplex Western blot experiment. Thepotential of antibody-oligonucleotide conjugates and complementaryoligonucleotide detectors in such an assay is evaluated. The protocol asshown schematically in FIG. 36 (left) wherein total protein from a celllysate is electrophoresed, transferred to a nitrocellulose membrane, anddetected in two steps by initially incubating the membrane with anantibody-oligonucleotide conjugate followed by incubation with acomplementary oligonucleotide-signal generator conjugate. As an example,the signal generator might be a horseradish peroxidase conjugate whichcan be localized on the blot by standard chemi-luminescent detection.This method was exemplified in a Western Blot assay using a human cancercell line, A431, that was untreated or treated with epidermal growthfactor (EGF). The cells were lysed and the protein fraction waselectrophoresed, transferred to a nitrocellulose membrane. As a controlexperiment, the cytoskeletal protein tubulin was detected either bystandard Western Blot conditions using a rat monoclonal anti-tubulinantibody followed by incubation with an anti-rat immunoglobulinsecondary antibody-HRP conjugate and developed using standardchemiluminescent methods. A separate blot was incubated successivelywith the same rat monoclonal anti-tubulin conjugated to HyLk1, washed,incubated with a HyLk1′-HRP conjugate and developed using standardchemiluminescent methods. The results documented in FIG. 36 (right) showthat both methods detect tubulin and a non-specific band to a similardegree with respect to sensitivity and specificity.

In certain embodiments, it will be advantageous to have a method wherebyvarious single antibody-oligonucleotide conjugates can be linked byhybridization to a choice of oligonucleotide-signal generatorconjugates. Such an application may allow a large catalog of antibodies,such as a library of monoclonal antibodies with different specificitiesto antigens, to be used together in multiplexed experiments. Here,assigning a single barcode to each antibody would be impractical.Instead, conjugating a single common oligonucleotide to each antibodywould be preferable. To accomplish this, as schematically represented inFIG. 37, a Universal Sequence (U) that can be conjugated to an antibodywas specified. Thereafter, a set of adapter oligonucleotides thatincorporate two sequences, one that hybridizes to the Universal Sequence(U′) and the second that is complementary to the sequence on anoligonucleotide-signal generator conjugate (A′, B′, etc.) may bedesigned. The complement to the Universal Sequence may be linked to thesequence that hybridizes to the signal generator via severalnon-hybridized bases or a polyethylene glycol chain or other non-nucleicacid hydrophilic linker such as a dendrimer to form individual adapters,e.g. U′-A′, U′-B′, and so on. Then, each antibody-Universal Sequenceconjugate would be mixed individually to an adapter and allowed tohybridize as in Step 1. Then, in Step 2, the antibody-Universal Sequenceconjugate/adapter complexes can be used individually or mixed togetherin an experiment to probe a biological sample. When the cognateoligonucleotide-signal generator conjugates are applied, as in Step 3,each hybridizes to the specific antibody carrying its complementaryadapter. As can be recognized, a panel of such adapter oligonucleotideseach comprising one sequence complementary to the Universal Sequence anda second sequence complementary to an oligonucleotide-signal generatorconjugate would allow a mix and match method to readily incorporate byhybridization a panel of different signal generators onto a panel ofantibody-Universal Sequence oligonucleotide conjugates.

In certain embodiments, a signal generator may be incorporated on the5′-end of the oligonucleotide-signal generator conjugate as shown inFIG. 38 or internally in the sequence.

In certain embodiments, an adapter can be used in an immunodection assayto allow a series of alternative signal generators to be used with asingle antibody-oligonucleotide conjugate. Here, two Western blotsidentical to those shown in FIG. 36 were probed for analysis by infraredimaging by a LI-COR instrument. In FIG. 39, the conventional approach ofusing an anti-rat immunoglobulin antibody conjugated to LI-COR IR800 dyeis shown in A. In B, the rat anti-tubulin-oligonucleotide conjugate wasinitially hybridized to an oligonucleotide designed to incorporateHyLk1′ and HyLk2 in tandem. This complex was then incubated with thenitrocellulose membrane and washed. Then, a HyLk2′-poly-IR800 dye signalgenerator conjugate was applied. LI-COR imaging reveals similarfluorescent detection of the tubulin band and the non-specificbackground band documents the results showing that the standard primaryantibody/secondary antibody-IR800 conjugate method and the UniversalAdapter method both detect tubulin.

The adapter method can also be used in flow cytometry as shown in FIG.40. Here as a positive control α-CD4 antibody-HyLk1 conjugate was addedto splenocytes, allowed to bind and washed. Subsequently, complementaryHyLk1′-poly-Dy490 was added, allowed to hybridize and detected by flowcytometry, which showed that 24% of the cells were CD4⁺ (A). In theadapter method, α-CD4 antibody-HyLk1 conjugate was added to lymphocytes,allowed to bind and washed. In (B), this was followed by addition of anadapter oligonucleotide designed to incorporate HyLk1′ and HyLk4 intandem. The adapter was allowed to hybridize to the HyLk1 conjugated toα-CD4 antibody and then washed. Subsequently, complementaryHyLk4′-poly-Dy649 was added allowed to hybridize and was detected byflow cytometry, which showed that 26% of the cells were CD4⁺ (B). In anegative control experiment (C), the α-CD4-HyLk1 probe was added tolymphocytes, then the HyLk1 ':HyLk4 adapter and then theHyLk1′-poly-Dy490 detector. If the adapter hybridizes, and therebyoccludes access of the detector to the α-CD4-HyLk1 probe, flow cytometrymay reveal no CD4⁺ cells.

In certain embodiments, multiple antibody-oligonucleotide conjugates maybe used simultaneously, or substantially simultaneously, to detect twoor more protein biomarkers in a multiplex bead array experiment. Here,the detection uses solid-phase sandwich immunoassays with matched pairsof antibodies, wherein a capture antibody tethers an antigen to asurface and the subsequent binding of a detector antibody thatrecognizes a distinct epitope on the antigen confirms detection. Themethod is schematically presented in FIG. 41 wherein (A) anantibody-oligonucleotide conjugate formed from a capture antibody isadded to a sample containing the antigen and allowed to bind, then (B)coded non-magnetic or magnetic beads conjugated to the complementaryoligonucleotide are added to the sample wherein theantigen/antibody-oligonucleotide complex hybridizes to the bead. Then,in (C), a biotinylated detector antibody is added and allowed to bindand in (D) streptavidin/R-phycoerythrin (SAPE) is added. The resultinglabeling of the bead by SAPE as in (E) allows the presence of antigen tobe detected by, for example, flow cytometry. In general, the relativeintensity of R-PE fluorescence will correspond to the relative abundanceof the antigen when multiple samples are compared by this assay.

FIG. 42 presents schematically the self assembly of multiplex beadarrays for analysis of multiple antigens in a single sample, as anextension of the principle described in FIG. 41. As an illustrativeexample, the diagram shows four antibody-oligonucleotide conjugatesassembling with four bead sets, such as non-magnetic or magneticfluorescently coded beads, conjugated to their respective complementaryoligonucleotides. Here, multiple antibody-oligonucleotide conjugates(Ab_(X)-HyLkX) might be added to a biological sample and each could bindits target antigen (Ag_(X)), as shown in (A). Thus, Ab₁-HyLk1 might bindAg₁, Ab₂-HyLk2 might bind Ag₂, and so on. Then, as in (B), a mixture ofmultiple sets of fluorescently coded beads, each bearing a differentoligonucleotide (HyLkX′-Bead_(X)), would be added. Using DNA directedassembly this allows self assembly of each antibody-oligonucleotideconjugate onto the cognate fluorescently coded bead as in (C). Then, notshown, addition of a mixture of biotinylated detector antibodiesspecific to each antigen, or biotinylated detector antibodies that mightdetect a common feature such as phosphorylation of tyrosine, to form asandwich complex, followed by washing, and then detection by SAPE andwashing, followed by analysis by, for example, flow cytometry. As such,the abundance of Ag₁ in the sample could be determined by the SAPEsignal associated with Bead₁, abundance of Ag₂ could be determined bythe SAPE signal associated with Bead₂, etc. Repeating this process withgreater numbers of matched pairs of antibodies and beads sets, and thenperforming the analysis on multiple samples may provide forsubstantially straightforward multiplexed quantitation of the relativeabundance of multiple antigens, or relative phosphorylation, or otherfeatures, in multiple samples.

In certain embodiments, antibody-oligonucleotide conjugates anddetectors comprising the complementary oligonucleotide conjugated to asignal generator, in this case a scaffold modified with multiplefluorophores, can be used in a multiplex bead array assay tosimultaneously, or substantially simultaneously, detect multipleanalytes from a sample. FIG. 43 schematically presents the methodwherein two antibody-oligonucleotide conjugates, Ab-HyLk1 and Ab-HyLk2,against a single protein target prepared from either two monoclonalantibodies to different epitopes on the target analyte that have beenconjugated to the two different oligonucleotides or from a polyclonalantibody that has been split into two parts and conjugated to the twodifferent oligonucleotides, then (A) the antibody-oligonucleotide pairs(comprising an antibody-oligonucleotide conjugate for detection and anantibody-oligonucleotide conjugate for capture) are added to a samplewherein the analyte is captured to form a sandwich immune complex, (B)fluorescently coded beads conjugated to the HyLk oligonucleotidecomplementary to the capture antibody-HyLk1 oligonucleotide conjugateare added to the sample wherein the sandwich immune complex hybridizesto the bead, then (C) a HyLk2′ complementary oligonucleotide-signalgenerator conjugate where the SG^(X) represents one or more signalgenerators, such as a biofluorescent protein such as R-PE or a polyfluorconjugate, is added and allowed to hybridize to the detectorantibody-HyLk2 oligonucleotide conjugate, tethering the fluorescentsignal generator to the bead as in (D), allowing steps of washing anddetection as, for example, by flow cytometry. As described, this schemepresents the method of a single-plex assay. However, by using the schemeas provided in FIG. 42, the design of the assay allows facile use ofmultiplexing with multiple sets of matched pairs for sandwichimmunoassay, via capture antibody-oligonucleotide conjugates hybridizingto their complementary fluorescently coded bead sets, and detection ofthe detector antibody-oligonucleotide complexes by hybridization tocomplementary oligonucleotides conjugated to fluorescence signalgenerators such as R-PE. In certain embodiments, the order of assemblyand addition to a sample might be varied, so that the capture antibodiesmight be combined with the beads prior to contact with the sample orafter, and the detector antibodies might be added to the sample prior toor along with the capture antibodies, or might be added after formingthe antigen-capture antibody complex on the beads and washing, or inother sequences as might be possible.

In certain embodiments, antigen-oligonucleotide conjugates andanti-isotype specific antibody-oligonucleotide conjugates and theircomplementary oligonucleotide conjugated to a signal generator, in thiscase a scaffold modified with multiple fluorophores, can be used in amultiplex bead array assay to simultaneously, or substantiallysimultaneously, detect and quantify antigen-specific antibodies such asauto-antibodies, antibodies generated from a vaccination or otherisotype specific antibodies such as IgE from a sample and detect andquantify the isotype response in a serology assay. FIG. 44 schematicallypresents a self-assembly-based method wherein (A) an antigen-HyLk1conjugate and an anti-isotype specific antibody-HyLk2 conjugate areadded to the blood sample wherein the anti-antigen antibody is capturedby both oligonucleotide conjugates and (B) added to mixture are HyLk1'-fluorescently distinct bead conjugates resulting in the capture of thecomplex by hybridization to HyLk1 conjugated to the antigen, (C) thecomplex is detected by the addition of a HyLk2′-signal generatorconjugate. As described, this scheme presents the method of a singleplex assay. However, the design of the assay allows facile use ofmultiplexing with multiple antigen-oligonucleotide conjugates and theircomplementary bead sets along with multiple anti-isotypeantibody-oligonucleotide complexes and complementary oligonucleotidesignal generators each conjugated to a different signal generator.However, in certain embodiments, the order of assembly and addition to asample might be varied, so that the capture antibodies might be combinedwith the beads prior to contact with the sample or after, and thedetector antibodies might be added to the sample prior to or along withthe capture antibodies, or might be added after forming theantigen-capture antibody complex on the beads and washing, or in othersequences as might be possible.

In certain embodiments, in a pre-assembly-based assayantigen-oligonucleotide conjugates may be hybridized to theircomplementary oligonucleotide immobilized on a bead or a bead encodedwith for example a fluorophore to be distinct from other beads, added tothe biological sample to bind the cognate antigen, washed and detectedwith an -antibody antibody (see exemplary FIG. 76). Example 24exemplifies the detection of a rabbit-BSA antibody that was captured bya BSA-HyLk1 conjugate pre-hybridized to a bead immobilized tocomplementary HyLk1′, followed by detection with a biotinylatedgoat—-rabbit antibody subsequently labeled with astreptavidin-phycoerythrin conjugate (SAPE) and analyzed by flowcytometry. The flow cytometry results (FIG. 75) presents the results ofthe capture and detection of -BSA antibody using BSA-HyLk1 conjugateimmobilized on Compel-HyLk1′ beads from 366 to 0.36 ng. However, incertain embodiments, the order of assembly and addition to a samplemight be varied, so that the capture antibodies might be combined withsample prior to addition of the beads, and the detector antibodies mightbe added to the sample prior to or along with the capture antibodies, ormight be added after forming the antigen-capture antibody complex on thebeads and washing, or in other sequences as might be possible.

In certain embodiments, it will be useful to determine the level and/ortype of immune response to antigens such as viruses, bacterialcarbohydrates, viral proteins, protein therapeutics.Antigen-oligonucleotide conjugates and their complementary detectors maybe used to simultaneously, or substantially simultaneously, detect forexample their cognate antibodies in a multiplex experiment to detect andtiter the amount of antibody present in a biological sample. This wouldallow for example multiplex detection and quantification of the level ofantibodies produced following immunization with a multiple antigen(e.g., combination) vaccine. FIG. 45 schematically presents theprocedure wherein (A) an antigen-HyLk1 oligonucleotide conjugate addedto a biological sample is bound by immunoglobulins present in thesample, (B) complementary HyLk1′ oligonucleotide conjugated onto afluorescently coded bead is added and theantigen-oligonucleotide/antibody complex hybridizes to the bead, (C) theantibody analyte is detected by addition of a biotinylatedanti-immunoglobulin detector antibody, (D) addition of astreptavidin/R-PE (SAPE) conjugate tethers the fluorescent detector tothe bead (E) to permit detection using, for example, a flow cytometer.In certain embodiments, the order of assembly and addition to a samplemight be varied, so that the capture antibodies might be combined withthe beads prior to contact with the sample or after, and the detectorantibodies might be added to the sample prior to or along with thecapture antibodies, or might be added after forming the antigen-captureantibody complex on the beads and washing, or in other sequences asmight be possible.

In certain embodiments, antigen-oligonucleotide conjugates and detectorscomprising the complementary oligonucleotide conjugated to a signalgenerator can be used in a multiplex bead array assay to simultaneously,or substantially simultaneously, detect multiple immunoglobulinspecificities from a single sample. FIG. 46 presents schematically theself assembly of multiplex bead arrays for analysis of multiple antibodyspecificities in a single sample, as an extension of the principledescribed in FIG. 45. As an illustrative example, the diagram shows fourantigen-oligonucleotide conjugates assembling with four bead sets, suchas non-magnetic or magnetic fluorescently coded beads, conjugated totheir respective complementary oligonucleotides. Here, multipleantigen-oligonucleotide conjugates (Ag_(X)-HyLkX) might be prepared sothat each could be bound by a different immunoglobulin as in (A). Then,as in (B), a mixture of multiple sets of fluorescently coded beads, eachbearing a different oligonucleotide (HyLkX′-Bead_(X)), would be added.Using DNA directed assembly this allows self assembly of eachantigen-oligonucleotide conjugate onto the cognate fluorescently codedbead as in (C). Then, not shown, these complexes could be added to aserum sample to allow binding of immunglobulins. After washing, additionof biotinylated detector anti-immunoglobulin antibodies to form asandwich complex, followed by washing, and then detection by SAPE andwashing, followed by analysis by, for example, flow cytometry. As such,the abundance of immunoglobulin reactive to Ag₁ in the sample could bedetermined by the SAPE signal associated with Bead₁, reactivity toAg_(e) could be determined by the SAPE signal associated with Bead₂,etc. Repeating this process with greater numbers of antigens and beadssets, and then performing the analysis on multiple samples may providefor substantially straightforward serology for multiple antigens inmultiple serum samples. In certain embodiments, the order of assemblyand addition to a sample might be varied, so that the capture antibodiesmight be combined with the beads prior to contact with the sample orafter, and the detector antibodies might be added to the sample prior toor along with the capture antibodies, or might be added after formingthe antigen-capture antibody complex on the beads and washing, or inother sequences as might be possible.

In certain embodiments, the principle of self-assembly directed byhybridization between pairs of complementary oligonucleotides can beused to facilitate the independent formation of multiple complexes. Thusin a bead array format, mixtures of antigen-oligonucleotide conjugatescan be used to detect and quantify one or more anti-antigen antibodiesin a serology assay and using pairs of anti-isotypicantibody-oligonucleotide conjugates with complementaryoligonucleotide-signal generators will be able to specifically detectand quantify the isotype response. However, in certain embodiments, theorder of assembly and addition to a sample might be varied, so that thecapture antibodies might be combined with the beads prior to contactwith the sample or after, and the detector antibodies might be added tothe sample prior to or along with the capture antibodies, or might beadded after forming the antigen-capture antibody complex on the beadsand washing, or in other sequences as might be possible.

The steps of self-assembly are illustrated in FIG. 47. Here (A)antigen-HyLk1 conjugate is added to the serum sample binding to itscognate antibodies, (B) coded beads immobilized with HyLk1′ is added tothe sample wherein the antibody/antigen-HyLk1 complex hybridizes to theHyLk1′ bead, (C) here for example an anti-IgG antibody conjugated toHyLk2 and an anti-IgE antibody conjugated to HyLk3 are added and allowedto bind to its their targets, (D) HyLk2′-SG¹ and HyLk3′-SG² fluorescentdetectors are added, allowed to hybridize and (E) detected by, forexample, a flow cytometer.

In certain embodiments, antibody-oligonucleotide conjugates and beadsconjugated to their respective complementary oligonucleotide can be usedin an immunoturbidity assay, which may be automated. FIG. 48schematically presents one iteration, according to certain embodiments,of this assay wherein two antibody-oligonucleotide conjugates against asingle protein target prepared from two monoclonal antibodies todifferent epitopes on the target analyte are conjugated to two differentoligonucleotides, Hylk3 and HyLk4, are added (A) to a sample containingthe cognate antigen, (B) allowed to bind, (C) combined with a mixture oftwo sets of the latex beads or gold particles immobilized to theirrespective, HyLk3′ or HyLk4′ complementary oligonucleotides, of a sizethat they are in suspension in hybridization buffer. As shown in (D) inFIG. 46, contact with the immune complex formed by the two antibodiesbinding as a sandwich to the antigen permits crosslinking between beadsby hybridization, leading to agglutination of the beads in the tube. Ingeneral, the degree of agglutination will be in proportion to theabundance of antigen, and is detected, for example, visually or using astandard immunoturbidity reader. In an alternate iteration a polyclonalantibody against a biomarker target is conjugated to an oligonucleotide.Also prepared is its complementary oligonucleotide immobilized on latexbeads or gold particles of a size that they are in suspension inhybridization buffer. These conjugates are processed as described hereinand the amount of agglutination may be determined on, for example, animmunotubidity reader to measure the abundance of the biomarker targetin the sample. The order of assembly and/or addition to a sample may bevaried, for example, so that the antibody-oligonucleotide conjugate(s)may be combined with the beads prior to contact with the sample orafter, or may be added after forming the antigen-capture antibodycomplex. FIG. 48 schematically presents the method wherein twoantibody-oligonucleotide conjugates against a single protein targetprepared from either two monoclonal antibodies to different epitopes onthe target analyte are conjugated to two different oligonucleotides,Hylk3 and HyLk4, or from a polyclonal antibody that has been split intotwo parts and conjugated to the two different oligonucleotides are added(A) to a sample containing the cognate antigen, (B) allowed to bind, (C)added to a tube containing two sets of latex beads, of a size that theyare in suspension in hybridization buffer, each conjugated to one of thetwo complementary oligonucleotides HyLk3′ or HyLk4′. As shown in (D),contact with the immune complex formed by the two antibodies binding asa sandwich to the antigen permits crosslinking among beads byhybridization, leading to agglutination of the beads in the tube. Ingeneral, the degree of agglutination will be in proportion to theabundance of antigen, and is detected, for example, using a standardimmunoturbidity reader. Here, for example, a polyclonalantibody that hasbeen raised against a biomarker target is divided and one half isconjugated to HyLk3 and one half is conjugated to HyLk4. Also preparedare two sets of beads conjugated to HyLk3′ and HyLk4′ respectively.These conjugates are processed as described herein and the amount ofagglutination may be determined on an immunotubidity reader to measurethe abundance of the biomarker target in the sample. However, in certainembodiments, the order of assembly and addition to a sample might bevaried, so that the capture antibodies might be combined with the beadsprior to contact with the sample or after, and the detector antibodiesmight be added to the sample prior to or along with the captureantibodies, or might be added after forming the antigen-capture antibodycomplex on the beads and washing, or in other sequences as might bepossible.

In certain embodiments, antibody-oligonucleotide conjugates anddetectors comprising the complementary oligonucleotide conjugated to asignal generator can be used in an ELISA-based assay using eitherfluorescent or enzyme-based detection. As presented in FIG. 49, (A)HyLk1′ oligonucleotides are immobilized by covalent attachment on asolid surface such as a plastic 96 well plate, and in the sample to beanalyzed, (B) a capture antibody-HyLk1 oligonucleotide conjugate bindsto its cognate antigen where present, (C) the sample is then added tothe oligonucleotide-coated surface and theantibody-oligonucleotide/antigen complex hybridizes to the HyLk1′,tethering the immune complex, (D) a biotinylated detector antibodyspecific to that antigen is added and (E) detected using astreptavidin-signal generator conjugate such as an enzyme orbiofluorescent protein. However, in certain embodiments, the order ofassembly and addition to a sample might be varied, so that the captureantibodies might be combined with the beads prior to contact with thesample or after, and the detector antibodies might be added to thesample prior to or along with the capture antibodies, or might be addedafter forming the antigen-capture antibody complex on the beads andwashing, or in other sequences as might be possible.

The method of FIG. 50 is similar to FIG. 49 except that theoligonucleotide is initially conjugated to a carrier protein such asbovine serum albumin (BSA) and thereby immobilized to the surface. Here,(A) oligonucleotides are immobilized on a solid surface such as aplastic 96 well plate but via covalent attachment or non-covalentadsorption of BSA-HyLk1′ conjugate, and in the sample to be analyzed,(B) a capture antibody-HyLk1 oligonucleotide conjugate binds to itscognate antigen where present, (C) the sample is then added to theBSA-HyLk1-coated surface and the antibody-oligonucleotide/antigencomplex hybridizes to HyLk1′, tethering the immune complex, (D) abiotinylated detector antibody specific to that antigen is added and (E)detected using a streptavidin-signal generator conjugate such as anenzyme or biofluorescent protein. Use of this indirect immobilizationmethod may be advantageous as: (i) the BSA may prevent non-specificbinding to the plastic surface, (ii) the attachment to a protein linkermay better present the oligonucleotide for hybridization (iii), lessoligonucleotide would be required as conjugation to protein is moreefficient than immobilization on plastic, and (iv) BSA will betteranchor the oligonucleotide to the plate by multi-point contact. Incertain embodiments, the order of assembly and addition to a samplemight be varied, so that the capture antibodies might be combined withthe beads prior to contact with the sample or after, and the detectorantibodies might be added to the sample prior to or along with thecapture antibodies, or might be added after forming the antigen-captureantibody complex on the beads and washing, or in other sequences asmight be possible.

In certain embodiments, antigen-oligonucleotide conjugates and detectorscomprising the complementary oligonucleotide conjugated to a signalgenerator can be used in an ELISA format in a serology-based assay todetect anti-antigen antibodies. FIG. 51 schematically presents aprotocol to accomplish this wherein an (A) a HyLk1′ complementaryoligonucleotide has been immobilized by attachment or adsorption of aBSA-HyLk1′ conjugate, (B) an antigen-oligonucleotide conjugate is mixedwith the biological sample capturing the anti-antigen immunoglobulin,(C) the resulting immune complex is captured by hybridization, (D) abiotinylated anti-antibody is added and (E) detected using astreptavidin-signal generator conjugated to an enzyme or biofluorescentprotein. Similar schemes may be used with complementary oligonucleotidesdirectly conjugated to the surface of the ELISA plate.

The bead-based examples illustrated herein may be applicable to bothself-assembly, i.e. wherein the capture-oligonucleotide conjugates areadded to the biological sample and then captured on beads, as well aspre-assembly, wherein the capture-oligonucleotide conjugate ispre-hybridized to its bead then mixtures of beads are combined and addedto the biological sample. In certain embodiments, the order of assemblyand addition to a sample might be varied, so that the capture antibodiesmight be combined with the beads prior to contact with the sample orafter, and the detector antibodies might be added to the sample prior toor along with the capture antibodies, or might be added after formingthe antigen-capture antibody complex on the beads and washing, or inother sequences as might be possible.

Immunocytochemistry

In certain embodiments, antigen-oligonucleotide conjugates and detectorscomprising the complementary oligonucleotide conjugated to a signalgenerator may be used in immunocytochemistry and related methods todetermine the abundance and localization of one or more specificantigens within cells. Where the signal generators can be distinguished,as is readily achieved by using fluorophores with distinct fluorescenceproperties, it would be straightforward to independently determine andthen compare the localizations of multiple antigens within a singlecell. After applying the probes and detectors, the sample would besubjected to microscopic imaging using optical means to illuminate thesample at each of the different fluorescence excitation bands and recordthe image at the corresponding emission bands, using sets of opticalfilters or other means. To determine the relative distributions, theimages could then be compared to registration images such as a phasecontrast or other brightfield image and then compared to each other toevaluate the distributions of intensity of fluorescence. Determinationof cellular abundance and distribution of antigens might commonly bepursued to examine cells grown in the laboratory to pursue anexperiment, or toward diagnosis, to examine cells obtained from abiological sample such as blood or other cell-containing fluid orextracted from a solid tissue as via a fine-needle biopsy, tissue printor other common method. As an example, as shown in FIG. 52 and FIG. 53,growing human cancer cells adhering to a glass surface werepermeabilized with a detergent, and fixed and dehydrated with methanol.The cells were rehydrated and incubated with BSA to block nonspecificbinding of probes and detectors. Then, a mixture of two probes, a ratanti-tubulin monoclonal antibody-HyLk1 conjugate and a mouseanti-phosphotyrosine monoclonal antibody-HyLk2 conjugate were applied.Then, the free probes were washed away and a mixture ofHyLk1′-poly-Dy490 and HyLk2′-poly-Dy549, both oligonucleotide conjugatesto fluorescently labeled amino dextrans, were applied. Then excessdetector conjugates were washed away and the cells were imaged byepifluorescence microscopy. As shown in FIG. 52, the characteristicdistribution of tubulin in cells can be appreciated, particularly inthose cells indicated by the white arrows that may be performingmitosis, a step in cell division where the microtubules align in thecell to mediate separation of chromosomes. In (A), both theanti-tubulin-oligonucleotide conjugate probe and complementaryoligonucleotide-fluorescence scaffold detector were applied. Here, oneinfers that the distribution of fluorescence in the image corresponds tothe distribution of tubulin in the cells insofar as the controlexperiment shown in (B), where only the fluorescent detector wasapplied, demonstrates a lower fluorescent signal and displays nosubcellular distribution.

Differential Interference Contrast Brightfield

In FIG. 53, in (A), a single field of cells is shown imaged byDifferential Interference Contrast brightfield, and in (B) and (C),respectively, the same field imaged using two fluorescent filter sets,the FITC filters to detect Dy490 distribution and the rhodamine filtersto detect Dy549 distribution. The three images allow independentevaluation and comparison of the shape of the cells, the distribution oftubulin and the distribution of phosphotyrosine-containing proteins. Theindependent nature of the detection can be appreciated at the positionindicated by the white arrows in each image. The arrows indicate twocells apparently performing mitosis, based on the pattern ofdistribution of tubulin. However, in the region of concentrated tubulinstaining, staining for phosphotyrosine appears to be absent.

Preassembly

FIG. 54 diagrammatically presents the process of preassembly using pairsof complementary oligonucleotides in which a plurality of molecularprobes, here a series of antibody-oligonucleotide conjugates, and aplurality of detectable components, here a series of signal generatingmoieties-complementary oligonucleotide conjugates, are hybridized, i.e.,preassembled, to form a plurality of preassembled molecularprobe-detectable component hybrids. The unique pairing of the molecularprobes and the detectable components, may be predefined, as in thisscheme, based on the complementarities of the oligonucleotidesconjugated to the binding moiety and signal generating moieties,respectively. In one embodiment, the preassembly process may becompleted by first mixing a plurality of antibody-oligonucleotideconjugates together to form a pool of the antibody-oligonucleotideconjugates, and then second, and separately, mixing a plurality ofsignal generating moiety-complementary oligonucleotide conjugatestogether to form a pool of the signal generating moiety-complementaryoligonucleotide conjugates. Subsequently, the pool of theantibody-oligonucleotide conjugates and the pool of the signalgenerating moiety-complementary oligonucleotide conjugates are thenmixed together, allowing for the hybridization of the complementaryoligonucleotide sequences to form the plurality of preassembledantibody-signal generating moiety hybrids. In certain embodiments, anindividual molecular probe, such as an individualantibody-oligonucleotide, may be combined and preassembled with itscomplementary detectable component comprising a complementaryoligonucleotide sequence, one at a time, each combination allowed topreassemble (i.e., hybridize). In certain embodiments, the individuallypreassembled molecular probe-detectable component hybrids may be mixedand pooled together. In certain embodiments, the preassembled molecularprobe-detectable component hybrids, either individual sets or aplurality of sets may then be brought into contact with a samplecomprising one or more molecular targets.

FIG. 55 presents results from an experiment demonstrating the process ofpreassembly as applied to a flow cytometry experiment on mousesplenocytes, and compares these results to sequential assembly in whichthe probes are applied in a first step and then the detectors in asecond step. In the four flow cytometry dot plots on the left (labeled“sequential 5-plex”), the experiment was performed according to Example10-B, wherein the CD4-HyLk1, CD8-HyLk2, CD19-HyLk3, CD43-HyLk4 andCD62L-HyLk5 antibody-oligonucleotide conjugates were applied to themouse splenocytes for 30 minutes at 4° C., followed by washing, and thenthe HyLk1′-Dy490, HyLk2′-Dy549, HyLk3′-Dy591, HyLk4′-Dy649 andHyLk5′-Dy405 detectors were applied 15 minutes at room temperature,followed by washing and flow cytometry. In the example on the right(labeled “Preassembled 5-plex (in pool)”), the CD4-HyLk1, CD8-HyLk2,CD19-HyLk3, CD43-HyLk4 and CD62L-HyLk5 were combined in equal amounts ina single tube to form a pool of antibody-oligonucleotide conjugates.Then, the complementary polyfluor signal generating moiety conjugatesHyLk1′-Dy490, HyLk2′-Dy549, HyLk3′-Dy591, HyLk4′-Dy649 and HyLk5′-Dy405were added in molar excess and allowed to hybridize for 15 minutes atroom temperature. The preassembled antibody-signal generating moietyhybrids were then added to mouse splenocytes as a pooled mixture,allowed to bind 30 minutes at 4° C., washed, and then analyzed by flowcytometry. Qualitatively similar results were obtained by each method,indicating that the order of assembly is not critical to the use ofnucleic acid hybridization to form hybrids between molecular probes anddetectable components to enable detection of multiple analytes, forexample multiple biological targets in a sample.

FIG. 56 presents results comparing the use of two alternative methods ofpreassembly. In the example on the left (labeled “Preassemble in pool,then add to cells”), the CD4-HyLk1, CD8-HyLk2, CD19-HyLk3, CD43-HyLk4and CD62L-HyLk5 were combined to form a pool, the complementarypolyfluor signal generating moiety conjugates HyLk1′-Dy490,HyLk2′-Dy549, HyLk3′-Dy591, HyLk4′-Dy649 and HyLk5′-Dy405 were added inmolar excess, incubated, and the mixture was then added to mousesplenocytes before analysis by flow cytometry as in FIG. 55. On theright (labeled “Preassemble one-by-one, pool, add to cells”), theCD4-HyLk1, CD8-HyLk2, CD19-HyLk3, CD43-HyLk4 and CD62L-HyLk5 were eachindividually combined with their complementary polyfluor signalgenerating moiety conjugates HyLk1′-Dy490, HyLk2′-Dy549, HyLk3′-Dy591,HyLk4′-Dy649 and HyLk5′-Dy405 in separate tubes, incubated to permitpreassembly hybridization, added individually to the splenocytes,allowed to bind 30 minutes at 4° C., washed, and then analyzed by flowcytometry. The comparable results of the two alternative methods ofpreassembly suggest that either these alternatives, or other preassemblyprotocols, may be followed with similar success.

The examples illustrated herein may be applicable to both self-assembly,i.e., wherein the capture-oligonucleotide conjugates are added to thebiological sample and then captured on beads, as well as pre-assembly,wherein the capture-oligonucleotide conjugate is pre-hybridized to itsbead then mixtures of beads are combined and added to the biologicalsample. In certain embodiments, the order of assembly and addition to asample might be varied, so that the capture antibodies might be combinedwith the beads prior to contact with the sample or after, and thedetector antibodies might be added to the sample prior to or along withthe capture antibodies, or might be added after forming theantigen-capture antibody complex on the beads and washing, or in othersequences as might be possible.

Cross-Talk

Several schemes, such as those exemplified in FIG. 57 may be consideredto address and/or decrease the potential for cross-talk, e.g.,non-specific labeling or background, such that a molecular probe may befalsely detected or obscured, due to a natural process ofoligonucleotide dissociation and hybridization. Some processes that maybe used to address the concern regarding cross-talk include, forexample, as illustrated in panel A, wherein the oligonucleotide sequenceof a non-hybridized molecular probe (i.e., not hybridized to acomplementary detectable component, may be hybridized with anunconjugated complementary oligonucleotide. Similarly, in the processillustrated in panel B, the complementary oligonucleotide sequence of anon-hybridized detectable component may be hybridized to an unconjugatedoligonucleotide. In other embodiments, such as those shown in panel C,the duplexes that are formed by preassembly hybridization of themolecular probe(s) and the detectable component(s) may be stabilized toprevent dissociation, for example, by the addition of natural orsynthetic minor groove binding agents, such as distamycin or Hoechst33258, or of natural or synthetic intercalating agents, such asdaunomycin or ethidium bromide.

Universal Oligonucleotide Sequence

In certain embodiments, one or more binding moieties, such as one ormore antibodies, may be conjugated to a universal oligonucleotidesequence, i.e., a single, common oligonucleotide that serves as auniversal tag, to provide one or more molecular probes differing in theidentity of the binding moiety conjugated to the universaloligonucleotide sequence. Similarly, one or more signal generatingmoieties, such as one or more scaffolds comprising one or more organicfluorophores, may be conjugated to a universal complementaryoligonucleotide sequence (i.e., an oligonucleotide sequencecomplementary that serves as a universal tag) to provide one or moredetectable components to facilitate detection in one or more channelsand/or formats, such as one or more fluorescent channels, detection viaone or more enzymes through enzymatic reactivity, or one or moreparticles. Using the process of preassembly, forming preassembledmolecular probe-detectable component hybrids by selecting the molecularprobes to be used and individually hybridizing these to selecteddetectable components in their own tubes, thereby forming the selectedpairings individually, provides a method that avoids or substantiallyavoids indiscriminate hybridization events between molecular probes anddetectable components that are all combined at once so that thespecificity would be lost via cross-talk. The individually preassembledhybrids of the process disclosed herein, may then be stabilized. Incertain embodiments, the stabilized preassembled hybrids may then bepooled together, and may then be subsequently contacted with a samplecomprising one or more molecular targets, to perform a multiplexedassay.

In FIG. 58 is diagrammed an example wherein a universal oligonucleotideis conjugated to a panel of molecular probes and a universaloligonucleotide complement is conjugated to a panel of signal generatingmoieties. For example, a panel of antibody-universal oligonucleotideconjugates are individually combined (i.e., preassembled) with a panelcomplementary universal oligonucleotide-signal generating moieties inseparate tubes to form preassembled antibody-signal generating moietyhybrids prior to stabilization. The preassembled hybrids may bestabilized and then may be contacted with a sample comprising one ormore molecular targets or analytes to perform an assay. In certainembodiments, as illustrated in FIG. 59, the stabilized preassembledhybrids may first be combined or mixed together to form a pool ofstabilized preassembled hybrids, and then the pool of stabilizedpreassembled hybrids may then be contacted with a sample comprising oneor more molecular targets or analytes to perform an assay.

The process of preassembly using a universal oligonucleotide and itscomplement has particular value in assembling assays on barcodedparticles, for example with flow cytometry-based multiplexedimmunodetection assays. As shown in FIG. 60, a panel of monoclonalantibodies (or other affinity agents that form the capture reagents forsandwich immunoassays) may be conjugated to a universal oligonucleotide.Similarly, a panel of barcoded particles may be conjugated to thecomplementary universal oligonucleotide. A multiplexed immunoassay arraymay be assembled by individually preassembling (combining) anantibody-universal conjugate, such as a monoclonal antibody-universalconjugate, with a barcoded particle, such as a bead-complementaryuniversal oligonucleotide conjugate, to allow for hybridization to forma preassembled antibody-bead hybrid. The preassembled antibody-beadhybrids may then be stabilized with unconjugated oligonucleotides orduplex stabilizers. The individual stabilized preassembled antibody-beadhybrids may then be contacted with a sample comprising one or moremolecular targets or analytes to perform one or more assays.Alternatively, as illustrated in FIG. 61, the individual stabilizedpreassembled antibody-bead hybrids may be combined with otherpreassembled sets of stabilized preassembled antibody-bead hybrids toform a pool, that may then be contacted with a sample comprising one ormore molecular targets or analytes to perform one or more assays. Oncethe one or more molecular targets or analytes are bound, the remainingunbound sample may be washed away and the one or more bound targets oranalytes may be detected, for example, by the addition of detectorreagents, such as polyclonal antibodies, or polyclonal antibodies in abiotinylated form. After washing away unbound detectors, binding of thebarcoded particles may be detected, for example by a streptavidin-basedprobe or other means.

Barcode

In certain embodiments, the detectable component may be a nucleic acid,such as an oligonucleotide, or a sequence thereof. Oligonucleotides maybe readily applied as detectable components if they include a sequencebarcode, called herein as “barcoded oligonucleotide detectablecomponents,” that may be recognized by either the binding of otherdetectable components, for example, a complementary oligonucleotidesequence conjugated to one or more fluorescent signal generatingmoieties or other signal generating moiety tags, or by hybridization toan array, or by sequence analysis, or by combinations or derivativesthereof. In certain embodiments, the barcoded oligonucleotide detectablecomponent may comprise a first oligonucleotide sequence, such as a20-oligonucleotide universal sequence, comprising a complementaryoligonucleotide sequence that permits hybridization to a universaloligonucleotide conjugated to a molecular probe, and a secondoligonucleotide sequence, such as a 20-oligonucleotide unique sequence,comprising a sequence that is unique to that detectable component, andoptionally other sequences that may be used to enable detection. Thebarcoded oligonucleotide detectable components may then be readilyadapted to the process of preassembly by assigning the individualsequences of the barcoded oligonucleotide detectable components toparticular molecular probes, as shown in FIG. 62. The hybrids may beindividually formed by selecting a molecular probe, combining it with abarcoded oligonucleotide detectable component, allowing forhybridization to occur, followed by stabilization of the preassembledmolecular probe-barcoded oligonucleotide detectable component hybrid. Incertain embodiments, the stabilized preassembled molecularprobe-barcoded oligonucleotide detectable component hybrids may bepooled, as shown in FIG. 63, and then contacted with a sample comprisingone or more molecular targets or analytes. After washing to removeunbound sample, the barcoded oligonucleotide detectable components thathave been retained and/or bound to the sample may then be assayed, forexample, by dissociating the hybridized barcoded oligonucleotidedetectable components away from their respective molecular probes, suchas by denaturing the duplexes. The de-hydybridized barcodedoligonucleotide detectable components may then be eluted and analyzed,either qualitatively for the presence of, or quantitatively for theabundance of, each barcoded oligonucleotide detectable component. Incertain embodiments, the process may be combined with a DNA sequencingtechnology, wherein such a combination may enable very high levels ofmultiplexing, such as tens, hundreds, or thousands, and may offeraccurate quantitation, broad dynamic range, low background and/or highspecificity.

Preassembly

As shown in FIG. 64, the process of preassembly is compatible with theuse of one or more universal adapters, comprising a common, universaloligonucleotide sequence that is complementary to a universaloligonucleotide sequence conjugated to a molecular probe and a uniqueoligonucleotide sequence that is complementary to a unique detectablecomponent, wherein the unique detectable component comprises a uniqueoligonucleotide sequence conjugated to a particular signal generatingmoiety. The one or more universal adapters may comprise 45-mersequences, wherein the universal sequence may be a 20-meroligonucleotide sequence, and the unique oligonucleotide sequence may bea 20-mer oligonucleotide sequence. In certain embodiments, one or moremolecular probes conjugated to universal oligonucleotides may bepreassembled with one or more universal adapters, comprising acomplementary universal oligonucleotide sequence and a uniqueoligonucleotide sequence, by individually mixing a particular molecularprobe with a particular universal adapter, thereby allowinghybridization to occur to form a preassembled molecular probe-universaladapter hybrid. In certain embodiments, the preassembled molecularprobe-universal adapter hybrid may be stabilized, may then be pooledwith the other the stabilized preassembled molecular probe-universaladapter hybrids. The pooled stabilized preassembled hybrids may then becontacted with a sample, comprising one or more molecular targets oranalytes, followed by binding of the one or more molecular targets withthe one or more pooled stabilized preassembled molecular probes. Incertain embodiments, one or more detectable components, comprising oneor more signal generating moieties conjugated to unique oligonucleotidesequences complementary to particular unique sequences of the one ormore universal adapters may be added to label the one or more boundtargets in the sample, thereby facilitating detection of the one or moretargets. In certain embodiments, this process may be used to combine theprocesses of preassembly and ordered assembly in cases where there mightbe advantages to both.

As diagrammed in FIG. 65, the detectable components may be designed tocomprise a first oligonucleotide sequence that is complementary to anoligonucleotide sequence of a molecular probe and a secondoligonucleotide sequence that comprises a sequence that has beenchemically modified with fluorescent moieties. In certain embodiments,the complementary oligonucleotide sequence of a molecular probe ishybridized, i.e., preassembled, with the first oligonucleotide sequenceof a detectable component to form a preassembled hybrid comprising annon-hybridized second oligonucleotide sequence that comprises a sequencethat has been chemically modified with fluorescent moieties. Thepreassembled hybrid may then be combined with an oligonucleotidesequence complementary to the second oligonucleotide sequence,comprising an oligonucleotide sequence modified with other fluorescentmoieties, thereby forming a hybridized ternary complex. In certainembodiments, the second oligonucleotide sequence may be labeled withfluorescent groups that can perform fluorescence resonance energytransfer (FRET) with fluorescent groups on the oligonucleotide sequencecomplementary to the second oligonucleotide sequence. FRET may result ina shift in fluorescent emission wavelength and/or to quenching offluorescence. In certain embodiments, the hybridization of the secondoligonucleotide sequence may be detected by a FRET signal, therebyallowing for a means of qualitatively, or possible quantitatively,detecting the presence of a specific molecular probe and/or theformation of a particular molecular probe-target complex.

In certain embodiments, one or more molecular probes may be preassembledwith one or more supports, such as a solid support, particle, or gelsupport, conjugated to oligonucleotide sequences complementary to themolecular probe oligonucleotide sequence. The supports may be but arenot limited to, for example, agarose or magnetic beads. In certainembodiments, the one or more molecular probes may be combined, as aplurality or individually with the one or more supports conjugated tocomplementary oligonucleotide sequences, followed by hybridization, toform a preassembled affinity matrix or material. The preassembledaffinity matrix or material may then be used, for example, to affinitycapture, purify, and then release one or more molecular targets, such asone or more biomolecular targets, each as a complex bound to theparticular molecular probe. For example, as diagrammed in FIG. 66, amolecular probe conjugated to a universal oligonucleotide sequence maybe combined with the complementary oligonucleotide conjugated to asupport to allow for preassembly hybridization and thereby form anaffinity matrix. In certain embodiments, the affinity matrix may then bewashed to remove free molecular probe. The affinity matrix may be, forexample, combined with a sample comprising one or more molecular targetsto enable binding and capture of the one or more targets. In certainembodiments, the unbound components of the sample may then be washedaway. In certain embodiments, it may be useful to use a displacementoligonucleotide or to denature the hybridization complex to release thebound target-molecular probe complex from the support for furtheranalysis. The displacement oligonucleotide may be, for example, anotheroligonucleotide sequence, an LNA, or a PNA, or combinations orderivatives thereof.

Alternatively, as diagrammed in FIG. 67, a molecular probe conjugated toa universal oligonucleotide may be combined with a sample comprising oneor more molecular targets to bind the one or more targets. The boundtarget-molecular probe complex may then be captured by a complementaryoligonucleotide conjugated to a support via hybridization. In certainembodiments, the other components of the sample may be washed away. Incertain embodiments, it may be useful to use a displacementoligonucleotide or to denature the hybridization complex to release thebound target-molecular probe complex from the support for furtheranalysis. The displacement oligonucleotide may be, for example, anotheroligonucleotide sequence, an LNA, or a PNA, or combinations orderivatives thereof. The examples illustrated herein may be applicableto both self-assembly, i.e. wherein the capture-oligonucleotideconjugates are added to the biological sample and then captured onbeads, as well as pre-assembly, wherein the capture-oligonucleotideconjugate is pre-hybridized to its bead then mixtures of beads arecombined and added to the biological sample. In certain embodiments, theorder of assembly and addition to a sample might be varied, so that thecapture antibodies might be combined with the beads prior to contactwith the sample or after, and the detector antibodies might be added tothe sample prior to or along with the capture antibodies, or might beadded after forming the antigen-capture antibody complex on the beadsand washing, or in other sequences as might be possible.

Minimizing Complexity for Antibody Labeling Choice

Certain embodiments are directed to methods and/or systems for reducingto a manageable proportions the number of catalog products a vendor oflabeled antibodies (or other bio-molecules used as binding detectors)must manufacture, stock, market, and/or distribute in order to fullysatisfy customers' needs for a substantially complete choice of labelalternatives for a substantial portion of the antibodies in the catalog.In certain aspects, a complete choice of label alternatives for anygiven antibody in the catalog.

Antibodies—biological proteins exhibiting high-affinity binding ofsingle target molecules—are widely employed throughout biologicalresearch, clinical diagnostics, pharmaceutical drug discovery, and otherdisciplines to enable immunoassays to detect and quantify molecules ofinterest (‘analytes’). Commonly, an antibody employed in immunoassaysmust be labeled with another molecule to render them detectable;frequently, the labels employed are fluorescent molecules (or ‘fluors’),which emit light over characteristic wavelength ranges (or ‘colors’).Approximately 36 different fluors are commercially available today asantibody labels, covering the color gamut from deep red to violet. In‘multiplexed’ assays, which aim to detect two or more different analytesin the same sample, two or more different antibodies are typicallyemployed together (for example, one for each analyte, although othervariations are possible), and typically the antibodies are labeled witha different colored fluor to enable them to be detected individually.Multiplexed assays are increasingly common in flow cytometry, where asmany as sixteen different analytes may be detected simultaneously orsubstantially simultaneously. This creates a need for antibodies labeledin a wide range of colors (i.e., at least 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, or more).

As it is conventionally practiced, labeling an antibody with a fluor isa highly skilled task beyond the means of most users, so antibodyvendors market antibodies pre-labeled with fluors. This presents thevendor with a ‘combinatorial explosion’ problem: offering X differentantibodies each labeled in Y different colors requires the vendor tomanufacture, stock, market, and distribute a total of X*Y individualproducts. For example, one leading vendor offers approximately 529different anti-human antibodies suitable for flow cytometry, labeledwith any of 16 different fluors, thus requiring 16*529=8,464 differentproducts in order to offer customers a complete selection (not countingproducts available in multiple unit sizes). Large numbers such as theseprove impractical in practice, so vendors commonly offer only a very fewof their most popular antibodies in a wide range of label colors, andoffer the rest of their antibodies labeled with typically no more thantwo or three colors (and frequently only one). This trade-off reducesthe complexity (in the case of one leading vendor reducing the productofferings to approximately 1,511) at the expense of reducing thelikelihood of being able to satisfy a users' need for a particularantibody labeled with a particular color (in the present example, 82% ofall possible antibody/color combinations are not available from oneleading vendor). Antibody vendors compete among themselves, in part, byoffering antibody/color combinations different from those of theircompetitors, so users frequently rely upon a combination of severalvendors to meet their full range of needs. Thus, a company which doesnot offer a full range of antibody/color combinations loses business toits competitors.

Certain disclosed embodiments make antibody labeling simple enough forusers to perform at point of use (see Example 22). In certainembodiments one or more antibodies are conjugated with one or more shortoligonucleotides A, and one or more fluors are conjugated with one ormore short complementary oligonucleotide A′. When such one or moreantibodies and such one or more fluors are mixed in solution, thecomplementary oligonucleotides bind one another, yielding one or morefluorescently-labeled antibody via formation of the A:A′ hybrid. Incertain embodiments, each antibody is conjugated with a shortoligonucleotide A, and each fluor is conjugated with a shortcomplementary oligonucleotide A′. When such an antibody and such a fluorare mixed in solution, the complementary oligonucleotides bind oneanother, yielding a fluorescently-labeled antibody via formation of theA:A′ hybrid.

Certain embodiments are directed to methods and/or systems that simplyand effectively reduce the complexity of vendors offering a completecollection of antibody/color combinations to their customers. Asubstantial portion or each of the X antibodies in the vendor's catalogis offered conjugated to oligonucleotide A, and a substantial portion oreach of the Y fluors in the vendor's catalog is offered conjugated tooligonucleotide A′. A customer or user requiring a given antibodylabeled with a given fluor then need only purchase the twooligonucleotide-conjugated products (one fluor and one antibody), andmix them at point of use. Thus, the vendor offers customers or users acomplete choice, or a substantially more complete choice, of thepossible antibody/color combinations while significantly reducing thecomplexity by offering X+Y products, as opposed to X*Y products. In theexample of the leading vendor's catalog discussed herein a total of16+529=545 products would offer customers a substantially complete orcomplete range of antibody/color choices while reducing the number ofproducts required by (8,464−545)/8,464=94%.

Currently, some vendors attempt to at least partially accommodatecustomers' needs for a wide choice of labels by offering antibodiesconjugated with biotin, a small molecule vitamin, and fluors conjugatedwith streptavidin, a bacterial protein which binds biotin with highaffinity. In principle, a customer can purchase a biotin-conjugatedantibody, a streptavidin-conjugated fluor, and mix these together tolabel any antibody with any fluor. Some of the disadvantages of thisconventional approach include one or more of the following:

-   -   Streptavidin is a relatively large protein (66 kDa in its active        tetrameric form), potentially presenting steric hindrance        problems in labeling analytes.    -   Streptavidin is a somewhat ‘sticky’ protein which may bind        non-specifically to sample components, producing high        backgrounds.    -   Because biotin is a common biological molecule, endogenous        biotin can cause background and specificity issues when        performing assays with certain biotin-rich tissues such as brain        and liver.    -   Samples containing endogenous biotin-binding proteins, such as        eggs or bacteria, pose specificity and background problems.    -   Harsh conditions are required to break the streptavidin-biotin        bonds in order to strip and re-probe samples.    -   Streptavidin-conjugated fluors are subject to proteolysis,        thermal denaturation, and other causes of product degradation    -   Because streptavidin is tetrameric (has four biotin binding        sites) it can crosslink multiple biotinylated antibodies

These disadvantages render the biotin/streptavidin technology anunfavored choice for most users. Another technology which is known inthe art involves a technology, in which IgG antibody can be labeled bythe user with a fluorophore-labeled Fab fragment directed against the Fcportion of that IgG. In practice, this technology has provenproblematic. The antibody/Fab complex is stable for only minutes.Additionally, this technology is applicable only to the labeling of IgGantibodies. Finally, fluorophore-conjugated Fab fragments are expensive,complicated, and time-consuming to produce.

In contrast, certain disclosed embodiments have one or more of thefollowing advantages. In certain aspects, the oligonucleotide hybrid isstable for an extended period of time. In certain aspects, thetechnology disclosed herein can be used to label a large range ofantibody isotypes. In certain aspects, the technology disclosed hereinis simple to use and/or more economical to manufacture. Theoligonucleotides employed, in certain aspects, are small (about 6 kDa)compared to streptavidin, and thus present less steric hindrance. Incertain aspects, smaller oligonucleotides demonstrate substantially lessstickiness and substantially little non-specific binding. In certainaspects, a properly chosen oligonucleotide sequence will not findendogenous complementary sequences in biological samples, or at leastreduce endogenous complementary sequences in biological samples, thusavoiding, or substantially reducing, higher background staining. Incertain aspects, relatively mild conditions can be employed todisassemble the oligonucleotide hybrids for strip and re-probetechniques. Generally, oligonucleotide-conjugated fluors are not subjectto significant proteolysis and/or significant thermal denaturation aspotential sources of product degradation. In certain aspects,complementary oligonucleotides hybrize in a strict 1:1 ratio;accordingly they are least likely to or cannot crosslink multipleantibodies.

Certain embodiments are directed to methods and/or systems comprising:i) a first series of molecular probes prepared by independentlyconjugating a first oligonucleotide sequence to at least 10 bindingmoieties; and ii) a second series of detectable components prepared byindependently conjugating a second oligonucleotide sequence to at least3 signal generating moieties, wherein the second oligonucleotidesequence is complementary to the first oligonucleotide sequence;wherein: a) an appropriate amount of a molecular probe from the firstseries may be mixed with an appropriate amount of a detectable componentfrom the second series to produce a hybridized molecularprobe-detectable component; and b) at least 90% of possible combinationsof said first and second series may be produced. In certain aspects, asubstantial portion, at least a majority, at least 60%, 70%, 75%, 80%,85%, 92%, 96%, 98%, 99% or 100% of possible combinations of said firstand second series may be produced. In certain aspects, the first seriesof molecular probes may be prepared by independently conjugating thefirst oligonucleotide sequence to at least 3 binding moieties, forexample, at least 5, 8, 15, 20, 25, 30, 50, 100, 150, 200, 250, 300,350, 400, 450, 500, 750, or at least 1000 binding moieties, or between3-10, 4-9, 5-12, 6-14, 7-16, 8-19, 9-20, 10-25, 12-30, 25-50, 40-70,50-80, 75-100, 80-125, 115-150, 130-200, 150-250, 175-300, 200-400,300-500, 350-750, 400-800, or 500-1000 binding moieties. In certainaspects, the second series of detectable components may be prepared byindependently conjugating the second oligonucleotide sequence to atleast 3 signal generating moieties, for example at least 2 signalgenerating moieties, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,17, 20, 25, or 30 signal generating moieties, or between 2-5, 3-5, 3-6,4-7, 4-9, 5-8, 5-10, 5-12, 6-14, 7-15, 8-13, 9-16, 10-17, 11-18, 12-19,15-20, 10-25, 12-30, or 25-50 signal generating moieties. Certainembodiments are directed to methods and/or systems for reducing tomanageable proportions the number of catalog products a vendor oflabeled molecular probes must manufacture, stock, market, anddistribute, comprising: i) a first series of customizable molecularprobes prepared by conjugating a first oligonucleotide sequence to atleast 10 binding moieties; and ii) a second series of customizabledetectable components prepared by conjugating a second oligonucleotidesequence to at least 3 signal generating moieties, wherein the secondoligonucleotide sequence is complementary to the first oligonucleotidesequence; wherein: a) an appropriate amount of a customized molecularprobe from the first series may be mixed with an appropriate amount of acustomized detectable component from the second series to produce ahybridized molecular probe-detectable component; and b) at least 90% ofpossible combinations of said first and second series may be produced.In certain aspects, a substantial portion, at least a majority, at least60%, 70%, 75%, 80%, 85%, 92%, 96%, 98%, 99% or 100% of possiblecombinations of said first and second series may be produced. In certainaspects, the first series of customizable molecular probes may beprepared by independently conjugating the first oligonucleotide sequenceto at least 3 binding moieties, for example, at least 5, 8, 15, 20, 25,30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, or at least1000 binding moieties, or between 3-10, 4-9, 5-12, 6-14, 7-16, 8-19,9-20, 10-25, 12-30, 25-50, 40-70, 50-80, 75-100, 80-125, 115-150,130-200, 150-250, 175-300, 200-400, 300-500, 350-750, 400-800, or500-1000 binding moieties. In certain aspects, the second series ofcustomizable detectable components may be prepared by independentlyconjugating the second oligonucleotide sequence to at least 3 signalgenerating moieties, for example at least 2 signal generating moieties,at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, or 30signal generating moieties, or between 2-5, 3-5, 3-6, 4-7, 4-9, 5-8,5-10, 5-12, 6-14, 7-15, 8-13, 9-16, 10-17, 11-18, 12-19, 15-20, 10-25,12-30, or 25-50 signal generating moieties.

Certain embodiments are directed to methods and/or systems comprising:i) a first series of molecular probes prepared by independentlyconjugating a first oligonucleotide sequence to at least 10 bindingmoieties; and ii) a second series of detectable components prepared byconjugating a second oligonucleotide sequence to at least 3 scaffoldshaving one or more signal generating moieties, wherein the secondoligonucleotide sequence is complementary to the first oligonucleotidesequence; wherein: a) an appropriate amount of a molecular probe fromthe first series may be mixed with an appropriate amount of a detectablecomponent from the second series to produce a hybridized molecularprobe-detectable component; and b) at least 90% of possible combinationsof said first and second series may be produced. In certain aspects, asubstantial portion, at least a majority, at least 60%, 70%, 75%, 80%,85%, 92%, 96%, 98%, 99% or 100% of possible combinations of said firstand second series may be produced. In certain aspects, the first seriesof molecular probes may be prepared by independently conjugating thefirst oligonucleotide sequence to at least 3 binding moieties, forexample, at least 5, 8, 15, 20, 25, 30, 50, 100, 150, 200, 250, 300,350, 400, 450, 500, 750, or at least 1000 binding moieties, or between3-10, 4-9, 5-12, 6-14, 7-16, 8-19, 9-20, 10-25, 12-30, 25-50, 40-70,50-80, 75-100, 80-125, 115-150, 130-200, 150-250, 175-300, 200-400,300-500, 350-750, 400-800, or 500-1000 binding moieties. In certainaspects, the second series of detectable components may be prepared byindependently conjugating the second oligonucleotide sequence to the oneor more scaffolds, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10scaffolds, or between 2-5, 3-6, 4-7, 5-8, 6-9, 7-10, or 8-15 scaffolds,wherein the one or more scaffolds comprises at least 3 signal generatingmoieties, for example at least 2 signal generating moieties, at least 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, or 30 signalgenerating moieties, or between 2-5, 3-5, 3-6, 4-7, 4-9, 5-8, 5-10,5-12, 6-14, 7-15, 8-13, 9-16, 10-17, 11-18, 12-19, 15-20, 10-25, 12-30,or 25-50 signal generating moieties. In certain aspects, the scaffoldsmay be selected from a bead, a dendrimer, a polysaccharide molecule, adextran, a protein, a peptide, a second oligonucleotide sequence, aportion of the oligonucleotide sequence that is not complementary to theoligonucleotide sequence of the molecular probe, a polymer, ahydrophilic polymer, a nanoparticle, or combinations or derivativesthereof.

Certain embodiments are directed to methods comprising: i) preparing afirst series of molecular probes by independently conjugating a firstoligonucleotide sequence to at least 10 binding moieties; and ii)preparing a second series of detectable components by independentlyconjugating a second oligonucleotide sequence to at least 3 signalgenerating moieties, wherein the second oligonucleotide sequence iscomplementary to the first oligonucleotide sequence; and iii) mixing,independently and in a matrix or semi-matrix fashion, an appropriateamount of a molecular probe from the first series with an appropriateamount of a detectable component from the second series to produce aplurality of hybridized molecular probe-detectable components; whereinat least 90% of possible combinations of said first and second seriesmay be produced. In certain aspects, a substantial portion, at least amajority, at least 60%, 70%, 75%, 80%, 85%, 92%, 96%, 98%, 99% or 100%of possible combinations of said first and second series may beproduced. In certain aspects, the first series of molecular probes maybe prepared by independently conjugating the first oligonucleotidesequence to at least 3 binding moieties, for example, at least 5, 8, 15,20, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, or atleast 1000 binding moieties, or between 3-10, 4-9, 5-12, 6-14, 7-16,8-19, 9-20, 10-25, 12-30, 25-50, 40-70, 50-80, 75-100, 80-125, 115-150,130-200, 150-250, 175-300, 200-400, 300-500, 350-750, 400-800, or500-1000 binding moieties. In certain aspects, the second series ofdetectable components may be prepared by independently conjugating thesecond oligonucleotide sequence to at least 3 signal generatingmoieties, for example at least 2 signal generating moieties, at least 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, or 30 signalgenerating moieties, or between 2-5, 3-5, 3-6, 4-7, 4-9, 5-8, 5-10,5-12, 6-14, 7-15, 8-13, 9-16, 10-17, 11-18, 12-19, 15-20, 10-25, 12-30,or 25-50 signal generating moieties.

Certain embodiments are to a catalog, comprising: i) a first series ofat least 10 customizable antibodies, comprising at least one firstoligonucleotide sequence conjugated to the antibodies; and ii) a secondseries of at least 3 customizable detectable components, comprising atleast one second oligonucleotide sequence conjugated to the detectablecomponents, wherein the second oligonucleotide sequence is complementaryto the first oligonucleotide sequence; wherein: a) an appropriate amountof a antibody from the first series may be mixed with an appropriateamount of a detectable component from the second series to produce ahybridized antibody-detectable component; and b) at least 90% ofpossible combinations of said first and second series may be produced.In certain aspects, a substantial portion, at least a majority, at least60%, 70%, 75%, 80%, 85%, 92%, 96%, 98%, 99% or 100% of possiblecombinations of said first and second series may be in the catalog. Incertain aspects, the first series of customizable antibodies may beprepared by independently conjugating the first oligonucleotide sequenceto at least 3 antibodies, for example, at least 5, 8, 15, 20, 25, 30,50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, or at least 1000antibodies, or between 3-10, 4-9, 5-12, 6-14, 7-16, 8-19, 9-20, 10-25,12-30, 25-50, 40-70, 50-80, 75-100, 80-125, 115-150, 130-200, 150-250,175-300, 200-400, 300-500, 350-750, 400-800, or 500-1000 antibodies. Incertain aspects, the second series of customizable detectable componentsmay be prepared by independently conjugating the second oligonucleotidesequence to at least 3 signal generating moieties, for example at least2 signal generating moieties, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 17, 20, 25, or 30 signal generating moieties, or between 2-5,3-5, 3-6, 4-7, 4-9, 5-8, 5-10, 5-12, 6-14, 7-15, 8-13, 9-16, 10-17,11-18, 12-19, 15-20, 10-25, 12-30, or 25-50 signal generating moieties

Certain embodiments are directed to methods and/or systems furthercomprising a third series of universal adapters prepared byindependently selecting and pairing a complementary firstoligonucleotide sequence segment to the first oligonucleotide sequenceof the first series with a complementary second oligonucleotide sequencesegment to the second oligonucleotide sequence of the second series.Certain embodiments are to a catalog, further comprising a third seriesof universal adapters prepared by independently selecting and pairing acomplementary first oligonucleotide sequence segment to the firstoligonucleotide sequence of the first series with a complementary secondoligonucleotide sequence segment to the second oligonucleotide sequenceof the second series. In certain aspects, at least 90% of possiblecombinations of said complementary first oligonucleotide sequencesegment and said complementary second oligonucleotide sequence segmentmay be produced. In certain aspects, a substantial portion, at least amajority, at least 60%, 70%, 75%, 80%, 85%, 92%, 96%, 98%, 99% or 100%of possible combinations of said complementary first oligonucleotidesequence segment and said complementary second oligonucleotide sequencesegment may be produced. In certain aspects, the third series ofuniversal adapters may be prepared by independently selecting andpairing at least 3 of the complementary first oligonucleotide sequencesegments with at least 3 said complementary second oligonucleotidesequence segments, for example, at least 5, 8, 15, 20, 25, 30, 50, 100,150, 200, 250, 300, 350, 400, 450, 500, 750, or at least 1000, orbetween 3-10, 4-9, 5-12, 6-14, 7-16, 8-19, 9-20, 10-25, 12-30, 25-50,40-70, 50-80, 75-100, 80-125, 115-150, 130-200, 150-250, 175-300,200-400, 300-500, 350-750, 400-800, or 500-1000, of the complementaryfirst oligonucleotide sequence segments may be prepared by independentlyselected and paired with at least 5, 8, 15, 20, 25, 30, 50, 100, 150,200, 250, 300, 350, 400, 450, 500, 750, or at least 1000, or between3-10, 4-9, 5-12, 6-14, 7-16, 8-19, 9-20, 10-25, 12-30, 25-50, 40-70,50-80, 75-100, 80-125, 115-150, 130-200, 150-250, 175-300, 200-400,300-500, 350-750, 400-800, or 500-1000, of the complementary secondoligonucleotide sequence segments.

Optimization of Antibody Degree of Labeling

Certain embodiments are directed to systems and/or methods foroptimization of antibody degree of labeling. Certain embodiments providesystems and/or methods that allow users to choose one or more optimaldegrees of labeling for one or more antibodies, thereby avoidingspillover errors in multiplexed immunoassays. For example, for use inmultiplexed flow cytometry.

Antibodies—biological proteins exhibiting high-affinity binding ofsingle target molecules—are widely employed throughout biologicalresearch, clinical diagnostics, pharmaceutical drug discovery, and otherdisciplines to enable immunoassays to detect and quantify molecules ofinterest (‘antigens’). Typically, an antibody employed in immunoassaysmust be labeled with another molecule to render them detectable;frequently, the labels employed are fluorescent molecules (or ‘fluors’),which emit light over characteristic wavelength ranges (or ‘colors’).Approximately 36 different fluors are commercially available today asantibody labels, covering the color gamut from deep red to violet. In‘multiplexed’ assays, which aim to detect two or more different analytesin the same sample, two or more different antibodies are typicallyemployed together, the two more different antibodies are labeled withdifferent colored fluors to allow them to be detected individually.Multiplexed assays are increasingly common in flow cytometry, wheremultiply different analytes may be detected in the assay.

A substantially number of the fluors used to label antibodies haverelatively broad emission spectra (the range of wavelengths over whichthey emit fluorescent light), so that in multiplexed studies employingantibodies labeled with, for example, two fluors (1 and 2) the flowcytometer detection channel dedicated to detection of fluor l's emissionmay also “see” a relatively small amount of the light emitted by fluor2. This is often referred to as spillover. Some flow cytometers provideso-called ‘compensation’ mechanisms to correct for this spillover eitherelectronically or via software, but compensation often lacks sufficientaccuracy and, in the case of a very bright signal from fluor 1 spillingover into a channel observing a comparatively dim signal from fluor 2the unavoidable consequence of compensation is often an increase in thecoefficient of variation of the fluor 2 signal, which presents as abroadening of the apparent intensity distribution of fluor 2's signal.This broadening of fluor 2's intensity distribution constitutes anartifact which it is desirable to avoid in many instances, as theartifact makes it difficult and sometime impossible, to determine thepercentage of cells in the sample which express the antigen reported bythe fluor 2-labeled antibody with sufficient accuracy. Certainembodiments of the present disclosure are directed to allowing flowcytometrists to optimize, or substantial optimize, improve or fine tunethe brightness of a first labeled antibody fluor 1's fluorescence tominimize, substantially minimize or reduce its spillover into thedetection channel for a second labeled antibody fluor2. This may also beaccomplished in assays that have 3, 4, 5, 6, 7, 8, 9, or more fluorsthat may be affected by the spillover of one or more other fluors.

Certain disclosed embodiments provide methods and/or systems that allowthe user to adjust the brightness of a primary-labeled antibody. Incertain embodiments one or more antibodies are conjugated with one ormore short oligonucleotides A, and one or more Certain disclosedembodiments provide methods and/or systems that allow the user to adjustthe brightness of a primary-labeled antibody. In certain embodiments oneor more antibodies are conjugated with one or more shortoligonucleotides A, and one or more fluors are conjugated with one ormore short complementary oligonucleotide A′. When such one or moreantibodies and such one or more fluors are mixed in solution, thecomplementary oligonucleotides bind one another, yielding one or morefluorescently-labeled antibody via formation of the A:A′ hybrid. Incertain embodiments fluors are conjugated with one or more shortcomplementary oligonucleotide A′. When such one or more antibodies andsuch one or more fluors are mixed in solution, the complementaryoligonucleotides bind one another, yielding one or morefluorescently-labeled antibody via formation of the A:A′ hybrid. Incertain embodiments, each antibody is conjugated with a shortoligonucleotide A, and each fluor is conjugated with a shortcomplementary oligonucleotide A′. When such an antibody and such a fluorare mixed in solution, the complementary oligonucleotides bind oneanother, yielding a fluorescently-labeled antibody via formation of theA:A′ hybrid.

Certain embodiments are directed to kits that can be used for adjustingthe brightness a fluorescently-labeled antibody. These kits make thisadjustment substantially easier for the user. For example, certainembodiments are directed to a kit comprising one or moreoligonucleotide-conjugated antibodies and two or more vials ofcomplementary oligonucleotide-conjugated fluors, wherein the two or moreoligonucleotide-conjugated fluors bear the same fluorescent reportermolecule, substantially the same fluorescent reporter molecule or asimilar fluorescent reporter molecule, but at two or more differentdegrees of labeling, or ‘brightness’ (for example without limitation,four fluors per conjugate in a first vial, and 8 fluors per conjugate ina second vial), thus providing ‘dimmer and ‘brighter’ labels for theend-user to choose from. In the example of the previous section, theuser might choose to minimize or even eliminate the c.v. broadeningartifact by labeling antibody fluor 1 with a dimmer label (lower degreeof labeling) and antibody fluor 2 with a brighter label (higher degreeof labeling). Due to the physics of fluorescence, spillover is rarelyreciprocal between two fluors, because many fluorophores' emissionspectra are asymmetrical.

Due, in part, to the ‘combinatorial explosion’ problem faced by labeledantibody vendors as discussed herein, vendors have encountereddifficulties in readily providing an antibody labeled in a variety ofdegrees of labeling. Consequently, rather than making all permutationsavailable or customizing particular antibodies with specified degrees oflabeling, the vendors must pre-determine what antibodies are to be madeavailable, and typically only with the highest feasible degree oflabeling. Thus, end-users are unable to systematically adjust degree oflabeling in order to minimize compensation artifacts. Instead, userssometimes may choose different labels where spillover is a problem,using an inherently dim fluor to minimize spillover. However manyantibodies are not commercially available labeled with a large varietyof possible fluors, so an appropriate dim fluor may not be available, inwhich case the user is forced to accept the compensation artifact.

In the prior art, the only practical means to optimize labelingbrightness (for example, to achieve dimmer labeling) is for the user toperform his own antibody labeling in-house (or to contract with a vendorfor custom labeling), adjusting the labeling protocol to yield a lowerdegree of labeling. This is a time-consuming and highly skilled taskwhich is beyond the ability of most users, and custom labeling contractsare expensive and have long lead times. Additionally, conventionalcovalent labeling protocols are difficult or impossible to controlsufficiently to achieve a pre-determined precise degree of labeling. Incontrast, certain kits, methods, and or systems of the presentdisclosure make it simple and quick for the user to achieve preciselythe degree of labeling required (as illustrated in Example 21).

Certain embodiments are directed to a tunable detection method, kitand/or system, comprising: i) a molecular probe prepared by conjugatinga first oligonucleotide sequence to a binding moiety; ii) a series ofdetectable components, comprising different amounts of a signalgenerating moiety conjugated to a second oligonucleotide sequence,wherein the different amounts of the signal generating moiety provides arange of intensities of the signal generated, and wherein the secondoligonucleotide sequence is complementary to the first oligonucleotidesequence; wherein: a) the intensity of the signal generated can be tunedover a range 1.25 to 2×, 1.5 to 3×, 2 to 4×, 1.25 to 1.75×, 2 to 6×, 3to 5×, 3 to 6×, or 2 to 10× by selecting the detectable component havinga greater or lesser intensity. In certain embodiments, the intensity ofthe signal generated can be tuned over a range extending from the limitof self-quenching to the intensity of a single signal generating moiety.

Certain embodiments are directed to a tunable detection method, kitand/or system, comprising: i) a molecular probe prepared by conjugatinga first oligonucleotide sequence to a binding moiety; ii) a series ofdetectable components, comprising different amounts of a signalgenerating moiety conjugated to a second oligonucleotide sequence,wherein the different amounts of the signal generating moiety provides arange of intensities of the signal generated, and wherein the secondoligonucleotide sequence is complementary to the first oligonucleotidesequence; wherein: a) the intensity of the signal generated from atarget-bound molecular probe that is hybridized to a detectablecomponent can be tuned over a range greater than an order of magnitudeby selecting the detectable component having a greater or lesserintensity.

Simplifying Development of Multiplexed Flow Cytometry Assays

Certain embodiments are directed to systems and/or methods forsimplifying development of multiplexed flow cytometry assays. Thesemethods and/or systems may also be used for simplifying othermultiplexed assays as well, for example immunohistochemistry,microarray-based assays, bead-based assays, immunosorbant assays, etc.Multiplexed flow cytometry assays employ cocktails of two or moreantibodies, wherein the two or more antibodies are typically labeledwith a different-colored fluorophore, to analyze, for example, thesub-populations of cells in a cell sample where one or more of thosesub-populations are distinguished from other sub-population(s) withinthe sample by the co-occurrence (or lack of co-occurrence) of two ormore protein markers on or in the cells of the sub-population ofinterest. In certain embodiments multiplexed flow cytometry assaysemploy cocktails of two or more antibodies, wherein the two or moreantibodies are each labeled with a different-colored fluorophore, toanalyze, for example, the sub-populations of cells in a cell samplewhere one or more of those sub-populations are distinguished from othersub-population(s) within the sample by the co-occurrence (or lack ofco-occurrence) of two or more protein markers on or in the cells of thesub-population of interest. As an example without limitation, considerthe following hypothetical cell sample comprised of (at least) threedifferent cell types, wherein the cell types are defined by thespecified collection of protein markers indicated in Table 10-A.

TABLE 10-A Marker X Marker Y Marker Z Cell Type 1 + + − Cell Type 2 +− + Cell Type 3 + + + Cell Type 4 − + +

Table 10-A shows a collection of protein markers identifying 4 distinctcell types in a hypothetical sample. “+” indicates the presence of theindicated marker on the indicated cell type, whereas “−” indicates itsabsence.

In the example, no single marker can enable the unambiguousidentification of the four cell types. Similarly, no combination of justtwo markers can identify the three cell types (because cell types 1 and3 are both X+/Y+, cell types 2 and 3 are both X+/Z+, and cell types 3and 4 are both Y+/Z+). Only the combination of the markers X, Y, and Zpermits a unique immunophenotype to be assigned to each of the four celltypes. It is therefore necessary to employ a cocktail of three distinctantibodies (Abs)—Ab-X, Ab-Y, and Ab-Z (which bind exclusively to markersX, Y, and Z, respectively)—in order to separately enumerate the numberof cells of each of these four cell types occurring in this sample.

Examples of such multiplexed flow cytometry assays are common in bloodanalysis (where a single blood sample may contain 5, 10, 15, 20 or moredifferent cell types), immunology (where 4, 5, 6, 7 or moredistinguishable cell types may be present in a sample), and stem cellresearch (where the total number of distinguishable cell types in asample still frequently remains undefined at present)

The example of Table 10-A assumes that the distinct immunophenotypes ofthe four cell types of interest has already been defined. In earlystages of research, the effort to define such distinguishingimmunophenotypes may require multiplexing a much larger number ofantibodies in order to identify distinguishing combinations. It has beenestimated that the human genome encodes approximately 1,000 differentcell surface proteins.

In order for the 3 antibodies in this example to be separatelydistinguished by a flow cytometer it is necessary to separately labelthem with 3 distinct fluors, (Fl-1, Fl-2, and Fl-3, respectively) whosefluorescence emission spectra do not substantially overlap.

Further to the present example, if Ab-X, Ab-Y, and Ab-Z are commerciallyavailable labeled with Fl-1, Fl-2, and Fl-3, respectively, then thecytometrist may conduct the assay by purchasing the three labeledreagents Ab-X:Fl-1, Ab-Y:Fl-2, and Ab-Z:Fl-3. In contrast, if (forexample) Ab-Y and Ab-Z are both commercially available only labeled withFl-2, then the assay cannot be performed using commercial reagents. Thissituation is not infrequent. A review of the labeled antibody offeringsof numerous commercial vendors indicates that the median number of fluorcolors in which a commercial primary-labeled antibody is available isaround 2.

There therefore exists a need for systems and/or methods for antibodiesto be quickly and easily labeled by cytometrists with the desired fluor(approximately 3 or more distinct fluors are used in flow cytometry), inorder to facilitate the development and conduct of multiplexed assays.Using certain disclosed embodiments this may be accomplished asdiscussed herein. One exemplary approached is discussed in Example 29.The advantages of using the disclosed embodiments include one or more ofthe following: time savings (as users avoid complicated planning tasksto determine which antibodies can be accommodated in which detectorchannels); flexibility (when a new antibody is added to an existingmultiplex panel it may be added in the available detector channelwithout further modification of the existing panel, since the colors aremade available by certain embodiments of the present disclosure); andthe ability to optimize panels by minimizing spectral spill-over from achannel measuring a high-abundance marker into a channel measuring alow-abundance marker.

Automation

Many assays employing antibodies or other probes to detect multipletargets or analytes within samples or complex samples are oftenperformed by skilled personnel who select the targets to beinvestigated, perform extensive manipulation of the sample and of theassay reagents, or combinations thereof to obtain useful data. Whenmultiple targets or analytes are examined in a single sample byperforming multiple assays in parallel without substantially reducingreliability and/or reproducibility, this has the potential to offer oneor more advantages such as in speed and/or confidence in results. Speedis obtained by increasing the number of targets examined insubstantially the same time. Confidence is increased by permitting theexperimentor to embed positive and/or negative controls that validatethe experiment. Nonetheless, while such multiplexed assays aredesirable, they are difficult to perform by comparison to simple assaysof single targets or analytes. One or more limitations of the currentmethods serve as barriers to development of multiplexed assays. Forexample, one or more of the following: the ability to label individualprobes with readily distinguishable detectors is particularly poorlymatched to this task; the common methods and chemistries for labelingprobes such as antibodies with detectors such as fluorescent groups mayrequire optimization of each reaction for each pair; a similar situationis also found to attach a probe such as an antibody to a particle. As aresult, except for assays that will be performed multiple times and/orwill be utilized by multiple experimentors, the effort to develop andvalidate a multiplexed assay is generally considered impractical.Alternatively, once a multiplexed assay panel is developed, it is notreadily amenable to adaptation. For example, adding and/or exchangingone antibody probe for another may lead to loss of reliability and/orreproducibility for the whole panel. Certain of the disclosedembodiments are directed to increase the number of assays that can beperformed simultaneously or substantially simultaneously and to addressone or more of these problems in the art and other problems addressedherein.

For example, panels of antibodies are often used in flow cytometry suchas in the use of flow cytometry to examine the relative abundance ofcells in a sample that bear distinct patterns of surface antigens, asmay be performed in immunophenotyping, by employing panels of antibodiesthat are labeled with fluorophores. These fluorophores are oftenselected so that one or more antibodies may be detected in a differentfluorescence channel and the one or more antibodies may need to becompared to different controls. Current methods for labeling antibodieswith fluorescent groups are poorly matched to facile development ofpanels. The modification of each antibody with each fluorescent group isrelatively inefficient and must be optimized. Further, the resultingfluorescent conjugate must typically be purified before use. Further,extensive prior experience is required to obtain reliable and/orreproducible results. Even then, the difficulty of designing andvalidating each panel creates a high barrier. Certain embodiments aredirected to creating such a panel of fluorescently labeled antibodies todetect multiple targets on cells with high reliability and/orreproducibility. This reduces the high barrier to development of newpanels and/or modification of existing panels.

Another example, as disclosed herein, is the use of bead arrays ormicroarrays as panels of immunoassays to examine multiple targets in asoluble sample. Also disclosed herein is the use of multiple antibodies,often called capture antibodies that will bind a particular solubletarget or analyte that may or may not be in a sample. Then, a secondantibody, the detector, may bind to another site on the target to form asandwich. Alternatively, a competitor target or analyte might bind tothe capture antibody left unbound. This second binding can then bedetected by fluorescent or other tags. A scanning device is then used torecord the results by matching. To multiplex this sandwich immunoassay,the one or more capture antibodies would be bound to a differentparticle type, distinguishable by one or more fluorescent or otherbarcode, or tethered to a distinct position on a surface. A scanningdevice may then be used to record the results by examining the bead typeor microarray position. Alternatively, multiple antigens might beselected as probes to determine the presence of antibodies, as in thedetection of humoral immune responses. Again, to perform a multiplexedexperiment, the one or more antigens may be bound to one or moredifferent particle types or positions in a surface array. Similarly,current methods are poorly matched to simple and straightforward coatingof particles or surfaces with antibodies or antigens. The attachment ofantibodies or antigens is unpredictable, typically requiringoptimization and then careful washing to remove unbound probe and/orblocking to passivate or otherwise decrease nonspecific binding ofuncoated surfaces of beads or arrays. Certain embodiments are directedto creating such a bead array or microarray bearing multiple differentantibody or antigen probes to detect multiple targets or analytes withhigh reliability and/or reproducibility. Similarly, this reduces thehigh barrier to development of new panels and/or modification ofexisting panels.

It is well appreciated that automated systems can be of great advantagein performing immunoassays, particularly if they are able to rapidly andreliably perform multiplexed assays based on panels of tests. Further,an automated system might be of particular advantage if it couldassemble panels in many combinations, allowing use of a wide range ofantibodies or antigens, matched to a broad collection of fluorescentgroups or fluorescent particles, and providing the panels on anas-needed basis. However, current methods, as described herein, arepoorly matched to automation where assembly of a multiplexed assay wouldrequire new combinations of one or more antibodies with differentfluorescent groups or of one or more antibodies and/or antigens withdifferent fluorescent particles. The inefficient chemistry currentlyused is incompatible with simple and rapid assembly of such pairs on anas-needed basis. As disclosed herein, certain embodiments are directedto applying automation to combine antibodies with fluorescent groups orto bind antibodies to beads and thereby obtain reliable and/orreproducible panels on demand. This results in great advantages ofmultiplexing for increased speed and/or greater confidence. Certainembodiments disclosed herein are directed to automated systems and takeinto account the design and/or operational features of such devices. Forexample, devices and/or systems that analyze flow cytometry samplesand/or bead arrays gain advantages from application of the methodsand/or systems described herein. Certain embodiments make the selectionof optimal combinations of antibody probes and/or fluorescent detectorsor fluorescent beads much easier. Thus, the operator need only definethe panel of targets to be assayed and the automated system may designand/or form the multiplexed panel. Furthermore, certain embodiments aredirected to automated systems that incorporate algorithms to allow theresults of a prior assay for one panel of targets to help choose thetargets to be tested in a subsequent panel of assays, thereby allowingthe system to perform a series of tests with or without directsupervision or input of the operator. The systems and/or methodsdisclosed herein permit an increase in the number of different targetsthat may be examined within one or more panels, and thereby performmultiple assays with high reliability and/or reproducibility. Thisprovides significant advantages. Certain embodiments are directed toincreasing the ability of an automated system able to match a muchlarger number of antibodies, antigens, other probes or combinationsthereof with individual or multiple members of a larger set offluorescent groups or set of fluorescent beads to create a much largerrange of multiplexed panels that may be performed with greater speed andhigher confidence.

The following examples are provided by way of illustration, and are notintended to be limiting of the present disclosure. While certainembodiments have been disclosed, it will be understood that each iscapable of further modification and that this application is intended tocover variations, uses, or adaptations of the disclosure embodiments.

EXAMPLES

The following examples and protocols are given as particular embodimentsof the disclosure and to demonstrate the advantages thereof. It isunderstood that the examples and protocols are given by way ofillustration and are not intended to limit the specification or theclaims that follow. Additional information is also found in the attachedSoluLink manual, entitled Antibody-Oligonucleotide All-in-OneConjugation Kit User Manual, Catalog No. A-9201-001.

The conjugation examples below include a (1) HyNic antibody modificationstep, (2) conversion of an amino-oligonucleotide to a4FB-oligonucleotide and (3) and conjugation step. Following are commonprocedures used in the Examples that follow.

Antibody-HyNic Modification: The antibody is exchanged into ModificationBuffer (100 mM phosphate, 150 mM NaCl, pH 7.4) and a solution of S-HyNicin anhydrous DMF (X equivalents as described below) are mixed andincubated at room temperature for 1.5 h. The HyNic-antibody is purifiedto remove excess modification reagent and simultaneously bufferexchanged into Conjugation Buffer (100 mM phosphate, 150 mM NaCl, pH6.0) using a Zeba desalting column (ThermoPierce, Rockford, Ill.).

4FB-oligonucleotide preparation: 3′- or 5′-amino-modifiedoligonucleotide is exchanged into Modification Buffer and theconcentration is adjusted between 0.2 and 0.5 OD/μL. To the volume ofamino-oligonucleotide is added a ½ volume of DMF followed by addition ofS-4FB (20 equivalents in DMF). The reaction is incubated at roomtemperature for 1.5 hours, diluted to 400 μL with Conjugation Buffer(100 mM phosphate, 150 mM NaCl, pH 6.0) and desalted using a 5K MWCOVivaspin diafiltration apparatus. The 4FB-modified oligonucleotide iswashed with Conjugation Buffer (3×400 μL), the OD/μL of the purifiedoligonucleotide is determined and used directly in the followingconjugation reaction.

HyNic-antibody/4FB-oligonucleotide conjugation: To the HyNic-antibody (1mol equiv) in conjugation buffer is added 4FB-oligonucleotide (3-5 equivas described in the experiments). To the reaction mixture is added1/10^(th) volume TurboLink Buffer (100 mM aniline, 100 mM phosphate and150 mM NaCl, pH 6.0. The reactions are incubated for 2 hours andpurified as described below.

The gel data in the Figures were run on 4-12% Novex Bis-tris gels(Invitrogen, Carlsbad, Calif.) using MOPS Running Buffer (Invitrogen).Samples were loaded using NuPage LDS Sample Buffer (Invitrogen) withoutDTT or heating prior to loading.

Gels were developed as indicated with Coomassie blue for visual proteindetection, Lumetein protein stain (Biotium, Hayward, Calif.) or DNA DNASilver Stain (GE Healthcare, Piscataway, N.J.).

Example 1

In this Example a polyclonal antibody (bovine IgG (bIgG)) and a mousemonoclonal antibody (anti-FITC monoclonal antibody; JacksonImmunoResearch (Chadds Ford, Pa.)) were modified at 4 mg/mL with S-HyNic(20 equivalents). Following desalting into Conjugation Buffer theHyNic-antibodies were treated with a 35mer 5′-4FB oligonucleotide (5equivalents). The conjugates were purified using USY-20 size exclusionUltrafiltration Units (Advantec MFS, Inc., Dublin, Calif.). The DNASilver stained PAGE results for conjugation to bIgG are presented inFIG. 1. The loading, stain and samples in each lane are:

Loading: 400 ng antibody

Visualization/stain: Sybr Gold stain

Lane 1. Marker

Lane 2. 4FB-35mer oligonucleotide

Lane 3. HyNic-Bovine IgG

Lane 4. Bovine IgG/4FB-35mer oligonucleotide crude

Lane 5. Bovine IgG/4FB-35mer oligonucleotide purified

The Lumetein stained PAGE results for conjugation to b-IgG are presentedin FIG. 2. As shown in the gel in FIG. 2, there is significantconversion of antibody to conjugate. Lane 4 presents the shift of theproduct band to higher molecular weight and minor amounts of startingantibody as compared to Lane 3. In that the sensitivity of Lumeteinfluorescent protein stain is 1 ng this result would indicate greaterthan 90% conversion of antibody to conjugate as 400 ng of antibody wereloaded in each lane. The loading, stain and samples in each lane are:

Loading: 400 ng antibody

Visualization/stain: Lumetein stain (Biotium; Hayward, Calif.)

Lane 1. Marker

Lane 2. 4FB-35mer oligonucleotide

Lane 3. HyNic-Bovine IgG

Lane 4. Bovine IgG/4FB-35mer oligonucleotide crude

Lane 5. Bovine IgG/4FB-35mer oligonucleotide purified

The Lumetein stained PAGE results for conjugation to anti-FITCmonoclonal antibody are presented in FIG. 3. No unconjugated antibody isseen in lanes 3, 4 and 5 therefore based on the efficiency of conversionof antibody to conjugate is greater than 95% based on the sensitivity ofthe Lumetein stain. The loading, stain and samples in each lane are:

Loading: 150 ng antibody

Visualization/stain: Lumetein stain

Lane 1. Marker

Lane 2. HyNic-MS anti-FITC 150 ng

Lane 3. MS anti-FITC/4FB-35mer oligonucleotidecrude 300 ng

Lane 4. MS anti-FITC/4FB-35mer oligonucleotide purified 300 ng

Lane 5. MS anti-FITC/4FB-35mer oligonucleotide purified 450 ng

The DNA Silver stained PAGE results for conjugation to anti-FITCmonoclonal antibody are presented in FIG. 4. Unconjugated oligo can beseen in both lanes 4 and 5 demonstrating the inefficiency in removingexcess oligonucleotide using the USY 20 diafiltration filter. Thesensitivity of DNA Silver Stain is ˜50 pg oligo.

The loading, stain and samples in each lane are:

Loading: 150 ng antibody

Visualization/stain: Lumetein stain

Lane 1. Marker

Lane 2. HyNic-MS anti-FITC 150 ng

Lane 3. MS anti-FITC/4FB-35mer oligonucleotide crude 300 ng

Lane 4. MS anti-FITC/4FB-35mer oligonucleotide purified 300 ng

Lane 5. MS anti-FITC/4FB-35mer oligonucleotide purified 450 ng

Example 2

This experiment compares purification of antibody-oligonucleotideconjugates by diafiltration and adsorbing the conjugate on aZinc-chelate modified magnetic bead, washing the beads with buffer toremove excess 4FB-oligonucleotide and eluting the conjugate from thebead with imidazole-based eluting buffer.

Crude conjugate mixture prepared in Example 1 was purified by either a100 kD MWCO Vivaspin diafiltration spin column or Zinc-magnetic-bead toremove free oligo:

-   -   (A) Diafiltration purification: Conjugate was diluted into PBS        (400 μL) placed in the diafiltration apparatus and concentrated.        The retentate was diluted with PBS and concentrated 3 more        times.    -   (B) Zinc-chelate-magnetic-bead purification: Added crude        conjugated antibody/oligo mixture to Zn SepFast Mag        (Biotoolmics, UK) and bind for 30-40 mM. The beads were washed        (0.4 mL) with 25 mM sodium phosphate, 300 mM sodium chloride,        0.05% Tween-20, pH 7.5 4 times. The conjugate was eluted from        the beads with 2 5 mM EDTA, 300 mM NaCl, 250 mM Imidazole, 75        ug/mL HIS-6 peptide, pH 6.0, 4 times. The purified conjugate was        exchanged into 10 mM sodium phosphate, 149 mM sodium chloride, 1        mM EDTA, 0.05% sodium azide, pH 7.2.

As shown in FIG. 5, loading 300 ng of antibody and developing with DNASilver stain demonstrated near quantitative removal of excessoligonucleotide by adsorbing Ab-oligonucleotide conjugate on Zincmagnetic beads followed by release as no excess oligo is present in Lane5 while oligo can be seen in Lane 4. The loading, stain and samples ineach lane are:

Loading 300 ng of antibody

Stain: DNA Silver stain

Lane 1. Marker

Lane 2. 4FB-34FB-35mer oligonucleotide

Lane 3. Bovine IgG/34FB-35mer oligonucleotidecrude

Lane 4. Bovine IgG/4FB-35mer oligonucleotide purified with Diafiltrationspin column 100K

Lane 5. Bovine IgG/4FB-35mer oligonucleotide purified Zinc-magnetic-bead

Based on the sensitivity of DNA Silver Stain greater than 98% of theexcess is removed using this method.

Example 3

This experiment was designed to determine the optimal number ofequivalents of 4FB-oligonucleotide to be reacted with 1 mol equivalentHyNic-antibody to yield greater than 90% conjugate. To that end a 46merand a 35mer 4FB oligonucleotide were added to HyNic-anti-FITC antibodyat both 3 and 5 mol equiv/mol antibody. The conjugates were purified byadsorption/desorption on Zn-magnetic beads as described in Example 2.The loading, stain and samples in each lane are:

Loading: 300 ng of antibody

Stain: DNA Silver stain

Lane 1. Marker

Lane 2. 4FB-46mer 4FB-oligonucleotide

Lane 3. 1:5 MS anti-FITC/4FB-46mer oligonucleotide crude

Lane 4. 1:5 MS anti-FITC/4FB-46mer oligonucleotide purified

Lane 5. 1:3 MS anti-FITC/4FB-46mer oligonucleotide crude

Lane 6. 1:3 MS anti-FITC/4FB-46mer oligonucleotide purified

Lane 7. 1:5 MS anti-FITC/4FB-35mer oligonucleotide crude

Lane 8. 1:5 MS anti-FITC/4FB-35mer oligonucleotide purified

Lane 9. 1:3 MS anti-FITC/4FB-35mer oligonucleotide crude

Lane 10. 1:3 MS anti-FITC/4FB-35mer oligonucleotide purified

The DNA Silver stained PAGE results are presented in FIG. 6, includecrude reaction and purified product samples demonstrating that 5equivalents yielded a conjugate with more oligonucleotides/antibody asdeduced by the darker bands in the samples where 5 equivalents ofoligonucleotide were added.

Example 4

This experiment was designed to determine the optimal number ofequivalents of S-HyNic to be added to the antibody at 1 mg/mL to yieldgreater than 90% conversion to conjugate. In one experiment bIgG wasreacted with 20×, 30×, 40× and 50× equivalents of S-HyNic and reactedwith 5 equivalents of a 46mer 4FB-oligonucleotide. The DNA Silverstained PAGE results are presented in FIG. 7, showing excellentconversion to conjugate in the reactions as evidenced by the dark bandsin each lane and as the number of equivalents of S-HyNic are increasedthe number of oligonucleotides/antibody increases as the conjugate bandspenetrate the gel less as the number of equivalents of S-HyNic increasesresulting in the conjugation of more oligonucleotides/antibody. Theloading, stain and samples in each lane are:

Loading 300 ng of antibody

Stain: DNA Silver stain

Lane 1. Marker

Lane 2. 4FB-35mer oligonucleotide

Lane 3. 20× Bovine IgG/4FB-46mer oligonucleotide crude

Lane 4. 20× Bovine IgG/4FB-46mer oligonucleotide purified

Lane 5. 30× Bovine IgG/4FB-46mer oligonucleotide crude

Lane 6. 30× Bovine IgG/4FB-46mer oligonucleotide purified

Lane 7. 40× Bovine IgG/4FB-46mer oligonucleotide crude

Lane 8. 40× Bovine IgG/4FB-46mer oligonucleotide purified

Lane 9. 50× Bovine IgG/4FB-46mer oligonucleotide crude

Lane 10. 50× Bovine IgG/4FB-46mer oligonucleotide purified

Example 5

To determine the effect of length of oligonucleotide on conjugationefficiency 5 mol equivalents of 19mer, 39mer, 40mer, 46mer and 60mer4FB-modified oligonucleotides were reacted with a anti-Fitc monoclonalantibody that had been modified with 30 equivalents S-HyNic at 1 mg/mLantibody concentration. The DNA Silver stained PAGE results of thepurified conjugates are presented in FIG. 8, showing equivalent banddensity in each lane indicating that 4FB-oligonucletodes for length19mer to 60mer conjugate with equal efficiency. The loading, stain andsamples in each lane are:

Loading 1.0 ug of antibody

Stain: Commassie blue

Lane 1. Marker

Lane 2. HyNic-MS anti-FITC

Lane 3. Purified MS anti-FITC/4FB 19mer 4FB oligonucleotide

Lane 4. Purified MS anti-FITC/4FB-35mer oligonucleotide

Lane 5. Purified MS anti-FITC/4FB-40mer oligonucleotide

Lane 6. Purified MS anti-FITC/4FB-40mer oligonucleotide

Lane 7. Purified MS anti-FITC/4FB-46mer oligonucleotide

Lane 8. Purified MS anti-FITC/4FB-60mer oligonucleotide

The yields of the reactions based on BCA Protein Assay (ThermoPierce,Rockford, Ill.) were 55%, 52%, 50%, 50%, 47% and 50% for the 19mer,39mer, 40mer, 46mer and 60mer 4FB-modified oligonucleotides conjugationsrespectively.

Example 6

this Example a polyclonal antibody (bovine IgG (bIgG)) and a mousemonoclonal antibody (anti-FITC monoclonal antibody; JacksonImmunoResearch (Chadds Ford, Pa.)) were modified at 4 mg/mL with S-HyNic(20 equivalents). Following desalting into Conjugation Buffer theHyNic-antibodies were treated with a 35mer 5′-4FB oligonucleotide (5equivalents). The conjugates were purified using USY-20 size exclusionUltrafiltration Units (Advantec MFS, Inc., Dublin, Calif.). The DNASilver stained PAGE results for conjugation to bIgG are presented inFIG. 1. The loading, stain and samples in each lane are: This examplepresents the preparation and purification of an oligonucleotide/antibodyconjugate using the optimized conditions as determined in the Examplesabove. In this experiment 40mer and 60 mer 5′-amino-oligonucleotides asshown in TABLE 1 were 4FB-modified and conjugated to an antibody thatwas reacted with 30 equivalents of S-HyNic at 1 mg/mL then purifiedusing the Zn-magnetic bead adsorption/desorption method.

TABLE 1 # Base # Pairs MW Ext Coeff Oligonucleotide Sequence Oligo-1 4012451.2 374000 5′-G ACT GAC GAA CCG CTT TGC CTG ACTGAT CGC TAA ATC GTG-NH₂ Oligo-2 60 18557.1 5502005′-TTG CAT CGC CCT TGG ACT ACG ACT GAC GAA CCG CTT TGC CTG ACT GAT CGCTAA ATC GTG-NH₂

First, a stock solution of bovine IgG (bIgG) 5 mg/mL in modificationbuffer (100 mM phosphate, 150 mM NaCl, pH 7.4; Sigma (St. Louis, Mo.))was prepared. bIgG stock solution (20 μL; 100 ug bIgG) was diluted withmodification buffer (80 μL) to prepare a 1 mg/mL solution and wasexchanged into modification buffer (using a 0.5 mL Zeba 7K Desaltingcolumns (ThermoPierce, Rockville, Ill.)) pre-equilibrated withmodification buffer. A stock solution of S-HyNic (1.0 mg dissolved inanhydrous DMF (200 μL); SoluLink Biosciences (San Diego, Calif.)) wasprepared. To the bIgG in modification buffer was added S-HyNic/DMFsolution (1.12 μL; 30 mol equivalents). The mixture was mixed thoroughlyby pipette and incubated at room temperature for 2.0 h. Using a 0.5 mLZeba column the reaction mixture was desalted and buffer exchanged intoconjugation buffer (100 mM phosphate, 150 mM NaCl, pH 6.0). ThisHyNic-antibody was used directly in the conjugation reaction.

A 3′-Amino-modified 40mer Oligo-1 (11.1 ODs; Eurogentec (San Diego,Calif.)) was dissolved in 50 mM NaOH (30 μL) and was buffer exchangedinto modification buffer using a 0.5 mL Zeba desalting columnpre-equilibrated in modification buffer. The OD/μL of the final oligosolution was determined to be 0.33 OD/μL. A stock solution of S-4FB (1.0mg; SoluLink Biosciences) in anhydrous DMF (25 μL) was prepared. To thedesalted oligo was added DMF (15 μL) followed by S-4FB/DMF solution (3.7μL; 20 mol equivalents). The reaction mixture was thoroughly mixed andallowed to incubate at room temperature for 2 h. The reaction mixturewas exchanged into conjugation buffer (100 mM phosphate, 150 mM NaCl, pH6.0) using a 0.5 mL Zeba desalting column pre-equilibrated withconjugation buffer and the OD/μL was determined. This prepared a 4FBmodified 5′-amino-modified oligonucleotide that was used directly in theconjugation step.

3′-4FB-40mer Oligo-1 (30.8 μL; 5 mol equivalents) was added followed byaddition of TurboLink™ Catalyst (14 μL ( 1/10 volume); 100 mM aniline,100 mM phosphate, 150 mM NaCl, pH 6.0). The reaction mixture wasincubated at room temperature for 2 hours.

The IMAC Zn SepFast MAG Media (120 μL of a 50% slurry, Biotoolmics, UK)was prepped by addition of the beads 1.5 mL microcentrifuge tube,magnetizing the beads on a magnetic stand and the supernatant wasremoved. The beads were washed three times with binding buffer (200 μL;100 mM phosphate, 150 mM NaCl; pH 6.0). Following removal of the finalwash the entire volume (˜110 μL) of the completedantibody-oligonucleotide conjugation reaction was added directly ontothe bead pellet. The reaction/bead mixture was carefully mixed byswirling with a pipette tip for 30 seconds. The beads were allowed tosettle for 15 min at room temperature (18-25° C.). The slurry was mixedagain by swirling and allowed to settle for an additional 15 min. Thetube was placed on a magnetic stand for 1 min to pellet the beads andthe supernatant was gently removed and discarded. The bead pellet waswashed three more times with 400 μL wash buffer discarding thesupernatant each time.

The conjugate was then eluted and removed from the beads by adding 50 μLbead elution buffer (300 mM imidazole, 300 mM NaCl, 50 mM EDTA, 70 μg/mL(83.3 μM) (His)₆ peptide to the bead pellet. The slurry was gently mixedby swirling with a pipette tip for 30 sec and incubate the settledslurry for 15 minutes mixing gently at 5 minute intervals. The tube wasplaced into the magnetic stand to allow the beads to pellet for 1 min.The supernatant containing the affinity purifiedantibody-oligonucleotide conjugate was transferred into a new 1.5 mLtube. The beads were eluted three more times with 50 μL elution bufferto obtain the maximum conjugate recovery. The combined eluants werebuffer exchanged into storage buffer (PBS, 1 mM EDTA). Oligonucleotideconcentration was determined spectrophotometrically by determining theconjugate's absorbance at 260 nm. Antibody concentration was determinedusing the BCA assay (ThermoPierce, Rockville, Ill.). Typical yields are30-50% based on protein BCA assay. The molar substitution ratio is2.0-2.5 oligonucleotides/antibody. The conjugates were further analyzedby gel electrophoresis using 12% Bis-Tris Gel (Invitrogen (Carlsbad,Calif.)) and visualized by UV-backshadowing followed by Coomassie Blueor DNA Silver Stain (GE HealthCare (Piscataway, N.J.)).

Example 7

Protocol for preparation of an antibody/oligonucleotide conjugate on asolid phase support (Prospective).

MAC Zn SepFast MAG Media (120 μL of a 50% slurry, Biotoolmics, UK) canbe prepped by addition of the beads 1.5 mL microcentrifuge tube,magnetizing the beads on a magnetic stand and the supernatant can beremoved. The beads can be washed three times with Binding Buffer (200μL; 100 mM phosphate, 150 mM NaCl; pH 6.0). Antibody (100 ug) in 100 μLin Binding Buffer is added to the beads. The antibody/bead mixture canbe carefully mixed by swirling with a pipette tip for 30 seconds. Thebeads can be allowed to settle for 15 min at room temperature (18-25°C.). The slurry can be mixed again by swirling and allowed to settle foran additional 15 min. The tube can be placed on a magnetic stand for 1min to pellet the beads and the supernatant can be gently removed anddiscarded. The bead pellet can be washed three more times with 400 μLModification Buffer discarding the supernatant each time. To the beadslurry can be added a 20 mg/mL solution sulfo-S-HyNic (20-50 molequivalents) in Modification Buffer. The beads can be swirled andallowed to incubate at room temperature for 2 h. The bead reactionmixture can be diluted to 400 μL with Conjugation Buffer swirled andallowed to stand for 15 min. The tube can be placed on a magnetic standfor 1 min to pellet the beads and the supernatant can be gently removedand discarded. The bead pellet can be washed three more times with 400μL Conjugation Buffer discarding the supernatant each time. To the beadscan be added 4FB-oligonucleotide (3-5 equivalents) and a 1/10 volume ofTurboLink buffer. The reaction mixture can be swirled and allowed toincubate at room temperature for 1-16 h. The tube can be placed on amagnetic stand for 1 min to pellet the beads and the supernatant can begently removed and discarded. The beads can be washed with 25 mM sodiumphosphate, 300 mM sodium chloride, 0.05% Tween-20, pH 7.5 for 4 times.The conjugate can be eluted from the beads with 2 5 mM EDTA, 300 mMNaCl, 250 mM Imidazole, 75 μg/mL HIS-6 peptide, pH 6.0, 4 times. Thepurified conjugate can be exchanged into 10 mM sodium phosphate, 149 mMsodium chloride, 1 mM EDTA, 0.05% sodium azide, pH 7.2.

Example 8

Protocol for Preparation and Purification of Protein/OligonucleotideConjugate (Prospective):

For example, a Streptavidin/oligonucleotide conjugate can be preparedand purified using the following protocol.

Step 1: To a solution of streptavidin (1000 μL of a 5 mg/mL solution;Roche Biosciences) in modification buffer can be added a solution ofS-4FB (9.7 μL of a 10 mg/mL solution in anhydrous DMF; 10 mol equiv.).The reaction mixture can be gently vortexed and allowed to stand at roomtemperature for 1.5 h. The reaction mixture can be desalted intoconjugation buffer using a 2 mL Zeba column pre-equilibrated withconjugation buffer.

Step 2: His-tag conjugation: To 4FB-streptavidin prepared in step 1 canbe added HyNic-Peg2-His6-NH₂ (SoluLink Biosciences; 4.2 μL of a 20 mg/mLsolution in conjugation buffer; 0.75 mol equivalent). The His6-StAvconjugate can be purified by adsorption of the conjugate using His-TagPurification Chelating Agarose Beads (Agarose Bead Technologies (Tampa,Fla.) followed by washing to remove unconjugated streptavidin. Theconjugate can be eluted off the beads using imidazole/EDTA buffer. Theisolated HyNic-Peg2-streptavidin conjugate can be desalted intoconjugation buffer using a 5 MWCO diafiltration apparatus to both desaltand remove unconjugated HyNic-Peg2-His6-NH₂.

Step 3: Preparation of HyNic-oligonucleotide: A 5′-amino-modified 38meroligonucleotide can be exchanged and concentrated into modificationbuffer (100 mM phosphate, 150 mM NaCl, pH 7.4) using a 5K MWCO Vivaspincolumn (Sartorius Stedim, Purchase, N.Y.). The final concentration canbe adjusted to 0.3 OD/. To the oligo in modification buffer (33.4 μL; 30nmol) is added DMF (16.7 μL) and S-HyNic (11 μL of a 10 mg/mL solutionin DMF; 15 equivalents; SoluLink Biosciences). The reaction mixture canbe vortexed and allowed to stand at room temperature for 1.5 hours). Thereaction mixture can be desalted into conjugation buffer (100 mMphosphate, 150 mM NaCl, pH 6.0) using a 5 KDa MWCO VivaSpin column.Resuspension into conjugation buffer and concentration can be repeated 3times. The oligo concentration can be adjusted to 0.25 OD/μL.

Step 4: Oligo conjugation and conjugate purification: To the4FB-StAv-His-tag conjugate in Conjugation Buffer prepared in Step 2 (1mol equivalent) can be added HyNic-38mer oligonucleotide (2.0 mol equiv)in conjugation and 1/10 volume TurboLink catalyst. The reaction mixturecan be incubated at room temperature for 2 hours and the 38meroligonucleotide-StAv-His-tag conjugate can be purified by addition ofthe reaction mixture to Zinc-His-tag magnetic beads and incubated for 30min to allow the conjugate to bind to the beads. The supernatant can beremoved and the buffer (0.4 mL) can be added to the beads and themixture can be gently mixed using a pipette, incubated for 5 min andsupernatant can be removed. This washing procedure can be repeated 3more times. The conjugate can be eluted from the beads by adding elutionbuffer (100 mM imidazole; EDTA; buffer) incubating for 15 minutes. Thesupernatant can be removed and collected in a separate tube. The elutionprocedure can be repeated three more times. The combined eluants can beexchanged into 5 mM EDTA, PBS using a 0.5 mL pre-equilibrated Zebacolumn.

Example 9

General reagents and buffers: Modification Buffer (MB)-100 mM phosphate,150 mM NaCl, pH 7.4: Conjugation Buffer (CB)-100 mM phosphate, 150 mMNaCl, pH 6.0.

General procedure to prepare 5′-4FB-oligonucleotides from 5′-amino-HyLkoligonucleotides (see Table 2): 5′-amino-HyLk oligonucleotides(synthesized at Eurogentec, San Diego, Calif.) were dissolved inModification Buffer (500 μL) and desalted using 5K MWCO VivaSpindiafiltration devices (SartoriusStedim, Purchase, N.Y.). Theoligonucleotides were diluted to 190-250 μmol/μL. A solution of S-4FB(38.11 mg) in anhydrous DMF (500 μL) was prepared. In a specificexample- to amino-HyLk2′ (50-150 ODs) in Modification Buffer was addedDMF (½ vol) followed by S-4FB/DMF solution containing 20 mol equiv.S-4FB. The reaction mixture was incubated at room temperature for 2 hdiluted to 500 μL with nuclease free water and concentrated and washedthree times with nuclease free water using 5K MWCO VivaSpindiafiltration devices. The concentration of the recoveredoligonucleotide was determined. The degree of 4FB incorporation wasdetermined by a colorimetric assay wherein the 4FB-oligonucleotides wereincubated with 2-hydrazinopyridine to form a chromophoricbis-arylhydrazone that absorbs at A345 with molar extinction coefficientof 24500. This was performed by adding 4FB-oligonucleotide (2 μL) to a50 μM solution of 2-hydrazinopyridine dihydrochloride in 100 mM MES, pH5.0 (18 μL; SigmaAldrich; St. Louis, Mo.) and incubated at 37° C. for 30min followed by determination of the A345 absorbance using a NanoDropspectrophotometer (ThermoFisher). The degree of modification wascalculated using the following formula (measured A350/measuredA280)/(hydrazone molar extinction coefficient (24500)/theoreticaloligonucleotide molar extinction coefficient). Here, the degree oflabeling was 0.67-0.96.

TABLE 2 HyLk Sequences HyLk-1 5′-amino-cctgcgtcgtttaaggaagtac HyLk-1′5′-amino-gtacttccttaaacgacgcagg HyLk-2 5′-amino-ggtccggtcataaagcgataagHyLk-2′ 5′-amino-cttatcgctttatgaccggacc HyLk-35′-amino-gtggaaagtggcaatcgtgaag HyLk-3′ 5′-amino-cttcacgattgccactttccacHyLk-4 5′-amino-gctgacatagagtgcgatac HyLk-4′5′-amino-gtatcgcactctatgtcagc HyLk-5 5′-amino-tgtgctcgtctctgcatactHyLk-5′ 5′-amino-agtatgcagagacgagcaca HyLk-65′-amino-atgtacgtgagatgcagcag HyLk-6′ 5′-amino-ctgctgcatctcacgtacat

Example 10

General procedure to conjugate 4FB-HyLk oligonucleotides toHyNic-modified antibodies: The following example is representative ofthe protocol used to prepare the antibody/oligonucleotide conjugatesused.

Step 1) S-HyNic/antibody modification: anti-GK1.5 was concentrated to ˜5mg/mL using a 30 kD MWCO VivaSpin diafiltration apparatus followed bydesalting into Modification Buffer using a 0.5 mL Zeba desalting device.Anti GK1.5 concentration was determined using the BCA protein assay(ThermoPierce). A solution of S-HyNic (8.4 mg/mL) in anhydrous DMF wasprepared. To anti-GK1.5 (0.5 mg; 167 μL of a 3 mg/mL solution) was addedS-HyNic/DMF solution (2.3 μL; 20 mol equiv). The reaction mixture wasincubated at room temperature for 2.5 hours then desalted intoConjugation Buffer using Zeba desalting columns. The concentration ofthe HyNic-modified protein was determined to be 2.45 mg/mL.

Step 2) 4FB-oligonucleotide/HyNic antibody conjugation: To the solutionof HyNic-anti-GK 1.5 prepared in step 1 (0.5 mg; 3.27 nmol) was added4FB-HyLk-1 (3 mol equiv) and incubated at room temperature overnight.The conjugate was purified by size exclusion chromatography using aSuperDex 200 column (GE HealthCare) eluting with 100 mM phosphate, 150mM NaCl, pH 7.2 at 0.5 mL/min. The protein concentration of theconjugate was determined to be 0.122 mg/mL using the BCA assay.

TABLE 3 below lists the conjugates prepared and their respective data:Conjugate Antibody Clone Isotype GK1.5 (α-CD4) - HyLk1 α-CD4 (ratanti-mouse) GK1.5 1gG2a 145-2C11 α-CD3e (hamster 145-2C11 1gG (α-CD3) -HyLk2 anti-mouse) 145-2C11 α-CD3e (hamster 145-2C11 1gG (α-CD3) - HyLk3anti-mouse) 2.43.1 (α-CD8) - HyLk1 α-CD8 (rat anti-mouse) 2.43.1 1gG2b2.43.1 (α-CD8) - HyLk2 α-CD8 (rat anti-mouse) 2.43.1 1gG2b 1D3(α-CD19) - HyLk3 α-CD19 (rat anti-mouse) 1D3 1gG1

Example 10-B

Pre-Assembly of Antibody-Oligonucleotide Conjugates to ComplementaryOligonucleotide-Dextran-Polyfluorophore Conjugates.

Oligonucleotide sequences are detailed in Example 9, Table 2.

Antibody clone and isotype information is detailed in Example 10, Table3.

Antibody:oligonucleotide labeling conjugates were prepared as describedin Example 10.

Complementary oligonucleotide-dextran-polyfluorochrome detectorconjugates were prepared as described in Example 10.

Antibody:oligonucleotide conjugates and complementaryoligonucleotide:dextran:polyfluorochrome conjugates matched to createpreassembled labeling constructs are shown in Table 3B.

TABLE 3B Conjugated used to prepare preassembled labeling constructs:Antibody-Oligonucleotide Oligonucleotide:Dextran:PolyfluorochromeLabeling Conjugate Detector Conjugate α-CD4:HyLk1 HyLk1′:Dextran:Dy490α-CD8:HyLk2 HyLk2′:Dextran:Dy549 α-CD19:HyLk3 HyLk3′:Dextran:Dy591α-CD43:HyLk4 HyLk4′:Dextran:Dy649 α-CD62L:HyLk5 HyLk5′:Dextran:Dy405

Preassembly procedure: Antibody-oligonucleotide labeling conjugates werealiquoted at 0.1 μg (=6.67 pmol) IgG/sample. Each conjugate wasdetermined by A260 assay to have a molar substitution ratio ofoligonucleotide/antibody of approximately 2.0, as described in Example6. Therefore, each sample of antibody conjugate contains 2.0×6.67=13.3pmol of conjugated oligonucleotide. Complementaryoligonucleotide-dextran-polyfluorochrome detector conjugates were addedat a 1:1 oligonucleotide ratio. Each detector conjugate was determinedto have a component ratio of 1 mol complementary oligonucleotide:1 moldextran:5 mol fluorochrome, as described in Example 6. Therefore onesample of preassembled labeling construct contains the followingcomponents: 6.67 pmol IgG, 13.3 pmol oligonucleotide; 13.3 pmolcomplementary oligonucleotide, 13.3 pmol dextran, 66.5 pmolfluorochrome. For constructs prepared singly—one labeling conjugatecombined with one detector conjugate—elements were mixed in 40 μL finalvolume of 1% BSA-DPBS buffer and incubated with rotation for 15 minutesat 24° C. For constructs prepared “in cocktail”, as a mixture of fivelabeling conjugates and five detector conjugates, elements were combinedin 200 μL final volume of 1% BSA-DPBS buffer and incubated with rotationfor 15 minutes at 24° C.

Cell staining by preassembled labeling constructs: Spleen from a C57BL/6normal mouse was processed into a single cell suspension, and red bloodcells were lysed by hypotonic solution. Splenic leukocytes werealiquoted at a concentration of 1.2×10⁶ cells/sample, washed once in abuffer of 1% BSA in 1× Ca- and Mg-free DPBS, and resuspended for 20minutes in 50 μL of αFcR hybridoma culture supernatant to blocknon-specific binding of antibody IgG to cells. Preassembled labelingconstructs were then added to blocked leukocytes. Singly preassembledconstructs were pooled and then added to cells; constructs assembled in“cocktail” were directly added to cells. For either method, final IgGconcentration was 0.5 μg/250 μL or 2 μg/mL. Cells were stained for 30minutes at 4° C., followed by one wash in 500 μL 1% BSA-DPBS to removeexcess labeling construct.

Flow cytometric analysis: Samples were analyzed using a BD LSRII flowcytometer equipped with lasers and optical detectors suitable forexcitation of Dyomics fluorochromes and capture of emission spectra.10,000 events were taken per sample. Cell debris andmacrophage/granulocyte populations were excluded by gating lymphocytesbased on cell size on FSC vs. SSC dot plots. Analysis of lympocytepopulation subsets CD4+, CD8+, CD19+, CD43+ and CD62L+ was performedusing FlowJo software (Tree Star, Inc). Results are presented in FIG.56.

Example 11

Biofluor-oligonucleotide conjugate synthesis: Biofluor-oligonucleotideconjugates were prepared using the following 3-step general procedure(1) biofluor modification with S-HyNic, (2) preparation of4FB-oligonucleotide by modification of an amino-oligonucleotide withS-4FB (see above general procedure) and (3) conjugation of theHyNic-biofluor to 4FB-oligonucleotide.

Step 1) Biofluor modification procedure: R-Phycoerythrin (Febico,Taiwan) was exchanged into Modification Buffer (100 mM phosphate, 150 mMNaCl, pH 7.4) by dialysis. To a solution of R-PE (0.20 mg; 35 μL of 5.6mg/mL solution) in Conjugation Buffer was added S-HyNic (0.77 μL of a6.0 mg/mL solution in anhydrous DMF; 20 mol equiv). The reaction mixturewas gently vortexed and allowed to react for 2.5 h at room temperature.HyNic-R-PE was purified by desalting on a 0.5 mL Zeba desalting column(ThermoPierce, Rockford Ill.) pre-equilibrated with Conjugation Buffer(100 mM phosphate, 150 mM NaCl, pH 6.0).

Step 3) Conjugate formation: To a solution of HyNic-R-PE (175 ug of a2.0 mg/mL solution in Conjugation Buffer) was added 4FB-HyLk2′ (15 ug ofa 0.32 OD/μL solution in Conjugation Buffer: 3 mol equiv) and TurboLinkbuffer (1.8 μL; 100 mM aniline, 100 mM phosphate, 150 mM NaCl, pH 6.0;Solulink Biosciences, San Diego, Calif.) The reaction mixture wasincubated at room temperature for 4 h at room temperature and at 4° C.for 16 h. The conjugate was purified by size exclusion chromatography ona SuperDex 200 column (GE HealthCare, Piscataway, N.J.) to remove excessoligonucleotide. The conjugate was characterized by gel electrophoresis.

TABLE 4 Biofluor-oligonucleotide conjugates made by above protocol:Biofluor Oligonucleotide R-PE HyLk-2′ APC HyLk-1′ PerCP HyLk-3′

Example 11-B

Following the protocol of Example 11, the following is a standardprocedure to prepare an oligonucleotide-tandem dye conjugate: Anoligonucleotide-biofluor protein, e.g. R-PE, crosslinked APC or PerCP,as prepared above is concentrated using a diafiltration apparatus to 1-2mg/mL biofluor concentration and buffer exchanged into ModificationBuffer. A 20 mg/mL solution of second dye, e.g. Cy2, Cy3, Cy 3.5, Cy5,Cy5.5, Cy 7, Dyomics dyes, or Alexa dyes, in anhydrous DMF is prepared.To a solution of the oligonucleotide-biofluor conjugate is added 10-30equivalents of dye to incorporate sufficient dye to quench the donorfluorescence nearly completely, i.e. degree of modification 4-8. Theresulting phycobiliprotein conjugate is excited at 488 nm and thefluorescence emission is compared to that of unmodified R-PE excited atthe same wavelength. Highly efficient energy transfer (>99%) occurs fromthe protein to the fluorescent dye.

Example 12-A

Complementary Oligonucleotide-dextran-polyfluorophore Preparation: Theprocedure reported below was designed to prepare 1/1oligonucleotide/amino-dextran conjugates by conjugating <0.5oligonucleotide/dextran and isolating the heterodimer by (1) removal ofexcess oligonucleotide by size exclusion chromatography followed by (2)removal of excess amino-dextran by ion exchange chromatography.Subsequently the 1/1 oligonucleotide/dextran conjugate was modified withdye-N-hydroxysuccinimide esters to incorporate the desired dyes/dextran.This method can be used with amino-dextrans of various molecular weightsand the level of dye incorporation can be increased with increasingamino-dextran molecular weight. Furthermore this method could be usedwith other polymeric or dendrimeric scaffolds such as PANAM dendrimers(SigmaAldrich).

Step 1) Amino-dextran desalting: 70 kD amino-dextran (14.3 mg;Invitrogen; Carlsbad, Calif.) was dissolved in modification buffer (1.0mL), vortexed and heated at 55° C. to complete dissolution of theamino-dextran. The solution was desalted into Modification Buffer usinga 5 mL Zeba desalting column (Thermo Pierce; Rockford, Ill.). Volumeafter desalting was 1.27 mL and theoretical recovery was 11.26 mg/mL.

Step 2A) HyNic incorporation on amino-dextran: To a solution ofamino-dextran in Modification Buffer (0.24 umol; 14.3 mg; 1.27 mL of a11.26 mg/mL solution) was treated with a solution of S-HyNic (11.32 μLof a 26.2 mg/mL solution in anhydrous DMF; 5 mol equiv) and the solutionwas incubated at room temperature for 2.5 h and desalted intoConjugation Buffer using a 5 mL Zeba desalting column using a 250 μLbuffer stacker. The volume after desalting was 1.65 mL and a theoreticalconcentration based on 100% recovery of 8.67 mg/mL.

Step 2B) HyNic incorporation quantification: A 100 mM solution of2-sulfo-benzaldehyde (2-SB;) SigmaAldrich; St. Louis, Mo.) in 100 mMMES, pH 6.0 was prepared. HyNic-dextran as prepared above (2 μL) wasadded to the 2-SB solution (18 μL). A blank reaction wherein water (2μL) was added to 2-SB (18 μL) was also prepared. The solutions wereincubated at 40° C. for 30 min followed by determination of theabsorbance of the solution at A345. The concentration of thechromophoric hydrazone product was determined using its extinctioncoefficient of 28000. The HyNic substitution ratio, i.e. the averagenumber of HyNic groups/dextran, was determined by dividing the hydrazoneconcentration by the dextran concentration. In this reaction the MSR was3.36.

Step 3A) To a solution of HyNic-dextran as prepared in 2A (3.0 mg; 43nmol) was added 4FB-HyLkX′-oligonucleotide (0.5 equiv) and the reactionwas incubated at room temperature for 15 h. To remove excessoligonucleotide from the conjugate the solutions were purified by sizeexclusion chromatography on a SuperDex 200 column (GE HealthCare) usingLoading Buffer (20 mM HEPES, 25 mM NaCl; pH 7.00) as eluant at 1 mg/mLflow rate. The initial % of the first peak was collected andconcentrated to <800 μL using 5K MWCO VivaSpin columns and theconjugates were diluted to 800 μL with Loading Buffer. UV spectra of theconjugates were nearly identical with respect to A350/A280 ratios. TheA350 absorbance measures the bis-arylhydrazone conjugate bond. Toisolate oligonucleotide/dextran conjugate the solutions were passedthrough Vivapure Q Mini H devices using Loading Buffer in two 400 μLaliquots. The filter devices were washed with Loading Buffer (2×400 μL)to remove free dextran. The HyLkX′-oligonucleotide/dextran conjugateswere eluted from the support with 90 mM, 450 mM, and 750 mM NaCl inLoading Buffer. Most of the conjugate eluted in the 450 and 750 mMfractions. These two, along with the 90 mM elution, were pooled toafford conjugate in 1200 μL total volume. The pools were concentrateddown to approximately 150 μL, split, and desalted into ModificationBuffer over two 0.5 mL Zebas with 30 μL stacker.

Step 3B) General example of fluorophore incorporation onoligonucleotide-amino-dextran heterodimer conjugates: A solution ofDy490 (1.0 mg; Dyomics, Germany) was dissolved in anhydrous DMF (100μL). To a solution of oligonucleotide-dextran heterodimer (1.06 mg; 14.1umol) in Modification Buffer was added Dy490/DMF solution (12 μL; 10.7mol equiv) and the reaction mixture was allowed to incubate for 2 h atroom temperature than overnight at 4° C. The reaction mixture wasdiluted with Modification Buffer (1 mL) and loaded into a 10 kD dialysiscassette (ThermoPierce) and dialyzed against PBS (700 mL) overnight anda further 700 mL for 6 h. TABLE 5: The followingoligonucleotide/dextran/dye conjugates were prepared by the abovemethod:

TABLE 5 Oligo Dye HyLk-1′ Dy-490 HyLk-1′ LI-COR 680LT HyLk-2′ LI-COR800CW HyLk-5′ Dy-405 HyLk-6′ Dy-681 HyLk-1′ Dy490 HyLk-2′ Dy549 HyLk-3′Dy591 HyLk-4′ Dy649 HyLk-5′ Dy405 HyLk-6′ Dy681

Example 12-B

Preparation of Oligonucleotide-Dextran-Polyfluors with increasingnumbers of fluors/dextran:

The following procedure was used to prepareoligonucleotide-dextran-polyfluors of increasing numbers offluors/dextran: Solutions of Dy490, Dy591 and Dy649 in anhydrous DMF (20mg/mL) were prepared. To solutions of 20mer HyLk1′/amino-dextranheterodimer (1.0 mg at 1.0 mg/mL based on dextran as determined usingthe resorcinol assay) in Modification Buffer as prepared in Example 12-A(Step 3A) were added aliquots of dyes as listed in Table 5-B. Thereactions were incubated at room temperature for 3 h and transferred toPierce Slide-A-Lyzer MINI Dialysis Units, 20K MWCO and dialyzed against0.1M sodium phosphate, 0.15M NaCl, pH 7.2 for 8 h and the buffer waschanged and dialysis was continued for 4 h. The final conjugates werediluted to 1.0 mL with buffer. The degree of fluor incorporation wasdetermined using a NanoDrop 1000 Spectrophotometer using each dye'srespective absorbance maximum and molar extinction coefficient. Resultsare present in Table 5-B. Equivalents added were calculated by directlydissolving the dye in the vial from the vendor. The vials may have beenoverfilled therefore more dye than expected was added to the reaction.

TABLE 5-B Dy490 Dy591 Dy649 equiv equiv equiv equiv equiv equiv addedincorp added incorp added incorp 5.10 3.55 9.38 2.88 4.00 3.29 8.50 5.6311.80 4.64 6.67 5.69 11.90 7.60 16.50 6.53 9.33 8.04 17.10 10.57 23.509.22 13.33 12.58 25.50 14.37 35.30 13.62 20.00 21.64

Flow Cytometer Procedure to test these conjugates: anti-CD4-HyLk1conjugate (oligo/antibody ratio 4/1) on splenocytes from a C57BL/6 mousewere used to test these conjugates as described in Example 13. FIG. 68presents the flow cytometric results of the testing of this panel ofconjugates. FIG. 68 illustrates the evaluation of complementaryoligonucleotide-dextran-polyfluor conjugate detectors with increasingnumber of fluors/dextran scaffold. The detectors were pre-assembled onα-CD4-HyLk1 antibodies, allowed to hybridize, added to cells, washed andfluorescence intensity of bound hybrid was detected by flow cytometry.

Example 13

Complementary-oligonucleotide-HRP Conjugate Preparation:

Step 1) HyNic-modification of HRP: To a solution of HRP previouslydesalted into Modification Buffer (8.4 mg; 1.16 mL of a 7.27 mg/mL) wasadded sulfo-S-HyNic (Solulink Biosciences; 200 μL of a 11.25 mg/mLsolution in Modification Buffer; 30 mol equivalents). The reactionmixture was gently vortexed then allowed to stand at room temperaturefor 2 h then at 4° C. for 16 h. The reaction mixture was desalted intoConjugation Buffer using a 2 mL Zeba column.

Step 2) HyNic-HRP/4FB-oligonucleotide conjugation: To a solution ofHyNic-HRP (113 μL of a 6.45 mg/mL solution in Conjugation Buffer; 0.73mg) was added 5′-4FB-HyLnk1′ (73.5 μL of a 0.215 OD/μL solution inConjugation Buffer) and aniline to a final concentration of 20 mM. Thereaction was incubated overnight at 4° C. and the HRP-HyLnk1′ conjugatewas isolated by size exclusion chromatography on a SuperDex 200 column(GE HealthCare) eluting with PBS. EDTA to a final concentration of 1%was added to the conjugate to protect against nucleases. The finalconcentration of the conjugate was determined by measuring theabsorbance at 403 nm and calculating the concentration using E1%, 403nm=17.2.

Example 14

Flow cytometry Procedure:

Antibodies used for conjugation to HyLk oligonucleotide sequences: α-CD4(GK1.5), α-CD8 (2.43.1), and α-CD19 (1D3) were used to differentiate Tand B cell populations. α-CD43 (S7) and α-CD62L (MEL-14), whichrecognize adhesion molecules, were used to define sub-populations ofthese cell types. These antibodies were conjugated to HyLk sequences aslisted in Table 3.

Cell staining for flow cytometry analysis: Spleen from a C57BL/6 mousewas processed into a single cell suspension, and red blood cells werelysed by hypotonic solution. Splenocytes were used at a concentration of0.3×10⁶ cells/tube, and washed once in Facs buffer which consist of PBSwith 0.2% BSA and 0.012% Sodium Azide. To block non-specific binding ofantibodies to FcαR, cells were first incubated with 20 μL of a 2.4G2hybridoma supernatant for 10 minutes at room temperature. Withoutwashing the cells, 10 μL of primary Ab:HyLk oligonucleotide conjugateswere added at their appropriate concentrations (Ab:HyLk pairs weretitrated and used at 0.1-1.0 μg/sample). Cells were stained for 30minutes at 4° C., and then washed two times with FACS buffer to removefree antibody. Complementary HyLkX′:Dyomics-dye conjugates were thenadded, using 10 μL of the appropriate dilutions (the HyLkX′-Dy-dyes weretitrated and used at 0.03-0.3 μg/sample). Cells were incubated for 15minutes in the dark, at room temperature, and then washed two times withFacs buffer. Cells were resuspended in 350 μL of Facs buffer and run onan LSRII flow cytometer. For staining analysis, the lymphocytepopulation was selected by cell size on FSC vs. SSC dot plots. Analysiswas performed using FlowJo software (Tree Star, Inc). Results arepresented in FIG. 32.

Example 15

Western Blot Procedure: Chemiluminescent Detection of Tubulin by WesternBlot Using HybriLink Antibody-Oligonucleotide Conjugates.

Methods:

Step 1) Cell culture and EGF-stimulation. A431 human epidermoidcarcinoma cells (ATCC) were cultured in Dulbecco's Modified EssentialMedium (High Glucose “DMEM-HI”, from HyClone). DMEM was supplementedwith 4 mM L-glutamine (Gemini BioProducts) and 10% fetal bovine serum(FBS, also from Gemini)

Cultures were grown to confluence (˜1.5×10⁷ cells); supplemented mediawas removed from cultures by pipette, monolayers washed once withsterile D-PBS 1× (Sigma-Aldrich), and serum-free “starvation” DMEM wasadded to cultures for 24 hours to ensure complete metabolism ofsupplemental constituents.

Following treatment, cultures were harvested by manual dissociation(cell scraper) and transferred to conical tubes, in which they werepelleted by centrifugation for 5 minutes at 2000 RPM.

Step 2) Cell lysis and SDS-PAGE sample preparation: Lysis buffer(“Phospho-Safe”, EMD) was supplemented with 1× each protease andphosphatase inhibitor cocktails, plus 5 mM EDTA (HALT™ by Pierce ProteinResearch Products.)

1 mL of ice-cold supplemented lysis buffer was added to each pellet, andcells were resuspended by vigorous pipetting for one minute. Suspensionswere incubated on ice for 20 minutes with brief vortexing at 5-minuteintervals. Lysates were clarified by high-speed centrifugation (16,500×gfor 10 minutes at 4° C.).

Clarified supernatants were transferred to new sample tubes and analiquot of the sample was analyzed for protein concentration bycolorimetric analysis at 562 nm (BCA Assay Kit, Pierce).

To prepare samples for electrophoresis (SDS-PAGE), a concentratedTris-glycerol buffer containing SDS as a denaturant, β-mercaptoethanol(BME) as a reducing agent, and Bromophenol Blue as a sample trackingdye, was diluted to 1× in clarified lysate. (All components obtainedfrom Sigma-Aldrich.) Samples were heated at 95° C. for 5 minutes.

Step 3) SDS-PAGE and Electrotransfer: 20 μg of protein lysate was addedin duplicate to lanes of a pre-cast polyacrylamide gel (7% Tris-Acetate,Novex™, Invitrogen.) 20 μg of untreated lysate was loaded side-by-sideas a control. Samples were electrophoresed for 60 minutes at 150 voltsin the presence of tris-acetate electrophoresis buffer (Invitrogen).

Gels were immobilized on PVDF membrane (Immobilon-P™, Millipore) byelectrotransfer at 30V for 2 hours. Successful transfer was confirmed byreversible protein stain (Memcode®, Pierce).

Step 4) Membrane Preparation: The membrane was immersed in 99% methanol(Ricca) for 15 seconds and dried on the benchtop for one hour to fixproteins. It was then rehydrated by brief methanol immersion followed bysoaking in ultrapure water for 2 minutes.

A membrane blocking solution of 1% BSA in 1× Tris-Buffered Saline plus0.05% Tween-20 detergent (TBS-T) was applied to the hydrated membranefor one hour at room temperature with gentle agitation. (BSA, UnitedStates Biological; TBS-T, prepared from components obtained fromSigma-Aldrich).

Excess blocking solution was subsequently washed away with 3×5 minuterinses of TBS-T. The sample membrane was cut into identical halves atthis point to facilitate comparison of antibody detection strategies.

Step 5) Antibody Detection of Immobilized Tubulin: Purified rat IgGantibody against tubulin was obtained from a commercial source(Millipore).

α-tubulin:HyLk1 conjugate was prepared as described in Example 10. Theconjugate was evaluated against unconjugated antibody control in aside-by-side Western blot comparison.

Blots were incubated in 2 μg/mL of either conjugated or unconjugatedα-tubulin for 1 hour in 5 mL of blocking buffer, at room temperaturewith gentle agitation. Excess antibody was washed away by 3×5 minuterinses of TBS-T.

Step 6) Chemiluminescent Visualization of Anti-Tubulin: Complementary‘secondary’ conjugates to the enzyme horseradish peroxidase (HRP) wasadded to each membrane for detection by a chemiluminescent substrate(SuperSignal West® Pico™ ECL assay kit, Pierce).

In the method of a standard western blot protocol, α-rat IgG:HRP (GEHealthcare) was added to the membrane strip previously incubated withthe unconjugated rat α-tubulin. For the HybriLink™ western blot,oligonucleotide 1′-HRP conjugate was added to the membrane previouslylabeled with α-tubulin-HyLk1′.

HRP-conjugates were added at 100 ng/mL in 20 mL of blocking buffer andincubated with membranes at room temperature with rotation. The standardα-Rat IgG:HRP was incubated according to manufacturer's protocol for onehour, while the HybriLink secondary was incubated only briefly (for 15minutes) in accordance with the goal of the project, which is to achievecomparable substrate detection in significantly less time thantraditional methods.

Excess secondary conjugate was washed away with 3×5 minute rinses ofTBS-T.

Chemiluminescent substrate was added to both membranes according tomanufacturer's specifications, for 5 minutes at room temperature. Excesssubstrate was blotted away with filter paper (Whatman) and blots werevisualized by exposure to autoradiography film (Phenix). Film wasprocessed using a Konica developer and converted to digital image by aCanon document scanner. Results are presented in FIG. 36.

Example 16

Adaptor Design: The adapter design is shown in FIG. 37 wherein theUniversal sequence is conjugated to the primary antibody at the 5′-endof the oligonucleotide. The Signal Generator (AG) is conjugated to the5′-end of the complementary oligonucleotide. The adapter's sequence isconstructed 5′- to 3′- to be complementary to the oligonucleotides onboth the antibody and the signal generator. As demonstrated thealternate adapters as shown in FIG. 38 in which the signal generator isconjugated to the 5′-end of the oligonucleotide can be used. Table 6below presents the adapters prepared and tested.

TABLE 6 HyLk-Universal Antibody-5′-cctgcgtcgtttaaggaagtac-3′Ab-U/HyLk2′-SGAntibody-5′-cctgcgtcgtttaaggaagtac-3′//SG- 5′-cttatcgctttatgaccggacc-3′Splint: U′- HyLk 25′-ggtccggtcataaagcgataatgTTAATTgtacttccttaaacgacgcagg-3′ Ab-U/HyLk3′-SGAntibody-5′-cctgcgtcgtttaaggaagtac-3′//SG-5′- cttcacgattgccactttccac-3′Splint: U′- HyLk 35′-gtggaaagtggcaatcgtgaagTTAATTgtacttccttaaacgacgcagg-3′ Ab-U/HyLk4′-SGAntibody-5′-cctgcgtcgtttaaggaagtac-3′//SG-5′- gtatcgcactctatgtcagc-3′Splint: U′/ HyLk 45′-gctgacatagqgtgcgatacTTAATTgtacttccttaaacgacgcagg-3′ Ab-U/HyLk5′-SGAntibody-5′-cctgcgtcgtttaaggaagtac-3′//SG-5′- agtatgcagagacgagcaca-3′Splint: U′- HyLk 55′-tgtgctcgtctctgcatactTTAATTgtacttccttaaacgacgcagg-3′ Ab-U/HyLk6′-SGAntibody-5′-cctgcgtcgtttaaggaagtac-3′//SG-5′- ctgctgcatctcacgtacat-3′Splint: U′- HyLk 6 5′-atgtacgtgatgcagcagTTAATTgtacttccttaaacgacgcagg-3′Table Notes: CODE: U = Universal; Ab = antibody; HyLk = oligonucleotidename; SG = signal generator; TTAATT = linker sequence

Example 17

Infrared Detection of Tubulin by Western Blot Using AntibodyOligonucleotide Conjugates, adapter oligonucleotides and complementaryoligonucleotide-poly-IR dye conjugates

Methods:

A. Cell culture and EGF-stimulation.

A431 human epidermoid carcinoma cells (ATCC) were cultured in Dulbecco'sModified Essential Medium (High Glucose “DMEM-HI”, from HyClone.) DMEMwas supplemented with 4 mM L-glutamine (Gemini BioProducts) and 10%fetal bovine serum (FBS, also from Gemini)

Cultures were grown to confluence (−1.5×10⁷ cells); supplemented mediawas removed from cultures by pipette, monolayers washed once withsterile D-PBS 1× (Sigma-Aldrich), and serum-free “starvation” DMEM wasadded to cultures for 24 hours to ensure complete metabolism ofsupplemental constituents.

Cultures were then treated with (or without) Epidermal Growth Factor(EGF, Invitrogen) to stimulate tyrosine phosphorylation throughout theproteome. EGF was added at 100 ng/mL in serum-free DMEM containing 1%bovine serum albumin (BSA, United States Biological) for 7.5 minutes.Mock cultures were incubated with serum-free media plus 1% BSA withoutEGF.

Following treatment, cultures were harvested by manual dissociation(cell scraper) and transferred to conical tubes, in which they werepelleted by centrifugation for 5 minutes at 2000 RPM.

B. Cell lysis and SDS-PAGE sample preparation.

Lysis buffer (“Phospho-Safe”, EMD) was supplemented with 1× eachprotease and phosphatase inhibitor cocktails, plus 5 mM EDTA (HALT™ byPierce Protein Research Products.)

1 mL of ice-cold supplemented lysis buffer was added to each pellet, andcells were resuspended by vigorous pipetting for one minute. Suspensionswere incubated on ice for 20 minutes with brief vortexing at 5-minuteintervals. Lysates were clarified by high-speed centrifugation (16,500×gfor 10 minutes at 4° C.).

Clarified supernatants were transferred to new sample tubes and analiquot of each sample (EGF-treated vs. untreated) was analyzed forprotein concentration by colorimetric analysis at 562 nm (BCA Assay Kit,Pierce).

To prepare samples for electrophoresis (SDS-PAGE), a concentratedTris-glycerol buffer containing SDS as a denaturant, β-mercaptoethanol(BME) as a reducing agent, and Bromophenol Blue as a sample trackingdye, was diluted to 1× in clarified lysate. (All components obtainedfrom Sigma-Aldrich.) Samples were heated at 95° C. for 5 minutes.

C. SDS-PAGE and Electrotransfer.

20 μg of protein from EGF-treated lysate was added in duplicate to lanesof a pre-cast polyacrylamide gel (7% Tris-Acetate, Novex™, Invitrogen.)20 μg of untreated lysate was loaded side-by-side as a control. Sampleswere electrophoresed for 60 minutes at 150 volts in the presence oftris-acetate electrophoresis buffer (Invitrogen).

Gels were immobilized on PVDF membrane (Immobilon-P™, Millipore) byelectrotransfer at 30V for 2 hours. Successful transfer was confirmed byreversible protein stain (Memcode®, Pierce).

D. Membrane Preparation.

The membrane was immersed in 99% methanol (Ricca) for 15 seconds anddried on the benchtop for one hour to fix proteins. It was thenrehydrated by brief methanol immersion followed by soaking in ultrapurewater for 2 minutes.

A membrane blocking solution of 1% BSA in 1× Tris-Buffered Saline plus0.05% Tween-20 detergent (TBS-T) was applied to the hydrated membranefor one hour at room temperature with gentle agitation. (BSA, UnitedStates Biological; TBS-T, prepared from components obtained fromSigma-Aldrich.)

Excess blocking solution was subsequently washed away with 3×5 minuterinses of TBS-T. The sample membrane was cut into identical halves atthis point to facilitate comparison of antibody detection strategies.

E. Antibody Detection of Immobilized Tubulin.

Purified tubulin antibody from rat α-tubulin, clone YL1/2) was obtainedfrom a commercial source (Millipore).

α-tubulin-HyLk1 conjugate was prepared as described in Example 10. Theα-tubulin-HyLk1 conjugate was evaluated against unconjugated antibodycontrol by side-by-side western blot comparison.

Blots were incubated in 2 μg/mL of either conjugated or unconjugatedα-tubulin for 1 hour at room temperature in 5 mL of blocking buffer,with gentle agitation. Excess antibody was washed away by 3×5 minuterinses of TBS-T.

F. Visualization of Tubulin using Infrared Dye-Conjugated SecondaryDetectors.

Infrared detection of tubulin at 800 nm was conducted using eitherunconjugated control antibodies and anti-host infrared secondarydetectors, or oligo-conjugated antibodies and oligo-infrared dyeconjugate secondary probes.

Procedure was conducted as described in (A)-(E), except that theblotting membranes used for infrared detection were blocked in aproprietary buffer (Odyssey® Blocking Buffer, LI-COR Biosciences) ratherthan in 1% BSA-TBST. The membranes were blocked for one hour at roomtemperature, regardless of buffer.

Tubulin antibodies were applied as described in (E).

Infrared-dye conjugated host IgG antibodies (LI-COR Bioscience) wereapplied to control membranes labeled by unconjugated antibodies. IR800dye (LI-COR Bioscience) conjugated to oligo sequence HyLk1′ was appliedto membranes labeled by α-tubulin-HyLk1.

Secondary conjugates were diluted at 1:10,000 in appropriate blockingbuffer, supplemented with 0.2% Tween and 0.02% SDS, per manufacturerprotocol. Blots were incubated for one hour at room temperature, andexcess detector was removed by 3 washes of TBS-T.

Imaging was conducted using the Odyssey® Infrared Imaging System (LI-CORBioscience).

G. Using Oligonucleotide Adapter Sequences to Visualize Tubulin UsingNonspecific Secondary Detectors.

Infrared detection of tubulin was conducted as described in Sections(A)-(G), except that the tubulin antibody used was conjugated to HyLk1rather than HyLk2.

Oligonucleotide sequence adapter [HyLk1′-HyLk2] was applied afterantibody labeling with α-tubulin-HyLk1. Adapter was diluted at 100 ng/mLin blocking buffer and incubated on the membrane for 15 minutes at roomtemperature. Excess oligo adapter was removed by 3×5 minute washes ofTBS-T.

Detector conjugate HyLk2′-poly-IR800CW was applied as described in (G).Labeled tubulin was imaged at 800 nm using the Odyssey® Infrared ImagingSystem. Results are presented in FIG. 39.

Example 18

Flow cytometry Using Oligonucleotide Adapters to Facilitate Staining ofAntibody-Oligo Conjugates by Non-Complementary Oligo-Dye Detectors.

Methods:

A. Splenic leukocyte sample preparation.

Spleen from a B6 mouse was processed into a single cell suspension.Erythrocytes were lysed by hypotonic solution. Leukocytes were countedand suspended overnight in a culture medium consisting of DMEM (HyClone)with 5% fetal bovine serum (Invitrogen), 1% HEPES (Sigma-Aldrich), 1%non-essential amino acids solution (Sigma-Aldrich), 1%penicillin/streptomycin antibiotic (Invitrogen), and 0.00033%β-mercaptoethanol (GE Healthcare).

Samples were prepared the following day at a density of 6.0×10⁵cells/mL. Culture medium was removed by washing 2× in FACS buffer (D-PBS1× with 0.2% BSA and 0.012% sodium azide). (Buffer components obtainedfrom Sigma-Aldrich.)

Throughout this procedure, wash steps were conducted by centrifugationof samples for 5 minutes at 2000×g, followed by resuspension in 5000 μLof FACS buffer.

B. Antibody labeling.

Antibody against T-cell co-receptor CD4 (clone GK1.5) was purified frommouse hybridoma supernatant and supplied in a buffer of dialyzed PBS(University of Chicago, Fitch Monoclonal Antibody Facility).

α-CD4 antibody-HyLk1 conjugate was prepared as described in Example12-A.

Prior to addition of α-CD4HyLk1 conjugate to leukocyte samples,non-specific binding of IgG to the Fcδ receptor was blocked by theaddition of 20 μL supernatant from an α-FcδR producing hybridomaculture, clone 2.4G2 (University of Chicago, Fitch Monoclonal AntibodyFacility.)

Antibody-oligonucleotide conjugate was then added at 1 μg/sample in 500μL of FACS buffer and incubated for 30 minutes at 4° C.

Excess antibody conjugate was removed by 2 washes of FACS buffer.

C. Use of oligonucleotide adapters to facilitate cell staining.

Samples to be stained by non-complementary HyLk4′:Dy649 were firstincubated with an oligonucleotide adapter of the structure[HyLk1′:TTAATT: HyLk4].

Forward (5′->3′) and reverse (3′->5′) adapters were used in a 1:1 ratioto ensure a variety of structural orientations of the free HyLk4sequence for detection by HyLk4′:Dy649.

The adapter was applied at 2 μg/sample in 100 μL of FACS buffer for 15minutes at room temperature.

Excess adapter was removed by 2 washes of FACS buffer.

D. Cell staining with oligonucleotide-poly-fluorochrome detectorconjugates.

Following incubation with or without adapters, the samples were stainedby oligonucleotide-poly-fluorochrome detector conjugates. Controlsamples were stained with complementary detector HyLk1′:Dy490.Adapter-modified samples were stained with non-complementary detectorHyLk4′:Dy649.

Detectors were added at 30 ng/sample in 100 μL FACS buffer and incubatedfor 15 minutes at room temperature in the dark.

Excess detector was removed by 2 washes of FACS buffer. Samples weretransferred to sterile, 12×75 mm tubes for flow cytometry (BD Falcon®)at a final suspension of approximately 3×10⁵ cells in 5000 μL FACSbuffer.

E. Analysis by flow cytometry.

Samples were analyzed using a FACScanto™ flow cytometer, raw data fileswere acquired with FACSDiva™ software (BD), and files were interpretedusing FlowJo software (TreeStar). Results are presented in FIG. 40.

Example 19

Immunocytochemical visualization of microtubules using HybriLink™oligonucleotide conjugates

Methods.

A. Cell culture.

Human epidermoid carcinoma cell line A431 (American Type CultureCollection) is an adherent cell line exhibiting normal microtubulefunction throughout the cell cycle.

Cells were cultured in a medium consisting of DMEM (HyClone) with 10%fetal bovine serum (Invitrogen) supplemented with 4 mM stabilizedL-glutamine (Gemini BioSciences).

After being grown to log phase, the culture monolayer was dissociated by0.25% trypsin—53 mM EDTA (Invitrogen) in Hank's balanced salt solution(HBSS, Invitrogen). After recovery of cells by centrifugation,approximately 1.8×10⁴ cells in 100 μL culture medium were added to eachwell of an optical-glass based, culture treated, black 96-well plate(Corning CoStar®).

Plated cells were incubated overnight to allow complete adherence to theglass surface.

B. Sample preparation for labeling.

After one wash to remove culture medium in Dulbecco's Ca- and Mg-freePBS (DPBS, Sigma-Aldrich), cells were prepared for antibody labeling.

Microtubules were stabilized by a 30 second extraction in a bufferconsisting of 80 mM PIPES, 5 mM EGTA, 1 mM MgCl₂, and 0.5% Triton-X-100(components from Sigma-Aldrich). Cells were washed once in DPBS, andfixed by 5 minute incubation in ice-cold methanol (Ricca Chemical).

Fixed cells were rehydrated by 3×5 minute washes of Tris-buffered salinewith 0.05% Tween®-20 (TBS-T, Sigma-Aldrich).

Non-specific binding of antibody was blocked by incubating for one hourin 1% BSA-DPBS at room temperature (BSA Fraction V, US Biological).

C. Target labeling with antibody.

To label microtubules, a rat IgG antibody against α- and β-tubulin(Millipore) was used. The antibody was conjugated to HyLk1 using thechemistry described in herein.

Cells were labeled by anti-tubulin-HyLk1 conjugate at a concentration of10 μg/mL in 1% BSA-DPBS for two hours at room temperature.

Excess antibody conjugate was removed by 3×5 minute washes of DPBS.

D. Antibody detection by oligonucleotide:dye conjugate.

Detector HyLk1′-poly-Dy490 prepared as described in Example 12-A wasapplied to anti-tubulin-HyLk1 labeled cells at a concentration of 2μg/mL in 1% BSA-DPBS for 30 minutes at room temperature in the dark.

After 3×5 minute washes of DPBS, cell nuclei were counterstained for 5minutes in a solution of 0.5 μg/mL DAPI (Sigma-Aldrich) and post-fixedfor 15 minutes in 10% neutral-buffered formalin (Sigma-Aldrich) topreserve dye stability for long-term sample viewing.

E. Observation.

Labeled microtubules were observed at 40× magnification with a FITCfilter set on an AxioVert 40 CFL fluorescence-DIC microscope (Zeiss).Whole cells were viewed at 40× magnification by brightfield DICmicroscopy. Images were acquired with AxioVision® software (Zeiss).Results are presented in FIGS. 52 and 53.

Example 20

Immunocytochemical visualization of microtubules using HybriLink™oligonucleotide conjugates.

Methods.

A. Cell culture. Human epidermoid carcinoma cell line A431 (AmericanType Culture Collection) is an adherent cell line exhibiting normalmicrotubule function throughout the cell cycle. Cells were cultured in amedium consisting of DMEM (HyClone) with 10% fetal bovine serum(Invitrogen) supplemented with 4 mM stabilized L-glutamine (GeminiBioSciences). After being grown to log phase, the culture monolayer wasdissociated by 0.25% trypsin-53 mM EDTA (Invitrogen) in Hank's balancedsalt solution (HBSS, Invitrogen). After recovery of cells bycentrifugation, approximately I.8×I 04 cells in I 00 f. 1L culturemedium were added to each well of an optical-glass based, culturetreated, black 96-well plate (Corning CoStar®). Plated cells wereincubated overnight to allow complete adherence to the glass surface.

B. Sample preparation for labeling. After one wash to remove culturemedium in Dulbecco's Ca- and Mg-free PBS (DPBS, Sigma-Aldrich), cellswere prepared for antibody labeling. Microtubules were stabilized by a30 second extraction in a buffer consisting of 80 mM PIPES, 5 mM EGTA, 1mM MgCh, and 0.5% Triton-X-100 (components from SigmaAldrich). Cellswere washed once in DPBS, and fixed by 5 minute incubation in ice-coldmethanol (Ricca Chemical). Fixed cells were rehydrated by 3×5 minutewashes of Tris-buffered saline with 0.05% Tween®-20 (TBS-T,Sigma-Aldrich). Non-specific binding of antibody was blocked byincubating for one hour in 1% BSADPBS at room temperature (BSA FractionV, US Biological).

C. Target labeling with antibody. To label microtubules, a rat IgGantibody against a- and ˜-tubulin (Millipore) was used. The antibody wasconjugated to HyLk 1 using the chemistry described in herein. Cells werelabeled by anti-tubulin-HyLk I conjugate at a concentration of 10 flg/mLin 1% BSA-DPBS for two hours at room temperature. Excess antibodyconjugate was removed by 3×5 minute washes of DPBS.

D. Antibody detection by oligonucleotide:dye conjugate. DetectorHyLk1′-poly-Dy490 prepared as described in Example 12 was applied toanti-tubulin-HyLk1 labeled cells at a concentration of 2 flg/mL in 1%BSA-DPBS for 30 minutes at room temperature in the dark. After 3×5minute washes of DPBS, cell nuclei were counterstained for 5 minutes ina solution of 0.5 ˜tg/mL DAPI (Sigma-Aldrich) and post-fixed for 15minutes in 10% neutralbuffered formalin (Sigma-Aldrich) to preserve dyestability for long-term sample viewing.

E. Observation. Labeled microtubules were observed at 40× magnificationwith a FITC filter set on an AxioVert 40 CFL fluorescence-Die microscope(Zeiss). Whole cells were viewed at 40× magnification by brightfield DICmicroscopy. Images were acquired with AxioVision® software (Zeiss).Results are presented in FIGS. 52 and 53.

Example 21

Prophetic Example of Brightness Tuning

In the development of a flow cytometry assay it is desired to detect andquantify two biological targets, Target A and Target B, employing twospectrally adjacent detection channels, Channel

A and Channel B, respectively, where Target A is highly abundant in thesample and Target B is of significantly lower abundance, such that whenemploying two maximally labeled antibodies, anti-A and anti-B, spilloverof the optical signal from anti-A into Channel B artifactually reducesthe detectability of Target B. Since the intensity of the signal fromanti-A is more than sufficient in this assay, reducing the degree oflabeling (number of fluorophores per antibody molecule) of anti-A—and,optionally, increasing the degree of labeling of anti-B—can improve thedetectability of Target B without significantly affecting thedetectability of Target A.

A collection of separately contained reagents is provided to determinethe degrees of labeling of anti-A and anti-B which best optimizes thedetectability of Target B without unacceptably reducing thedetectability of Target A. This collection comprises a tube of anti-Aconjugated to an oligonucleotide, a tube of anti-B conjugated to thesame oligonucleotide, three tubes each containing the complementaryoligonucleotide conjugated to two, six, or ten Pacific Blue fluorophoremoieties (PB-2, PB-6, and PB-10, respectively), and three tubes eachcontaining the complementary oligonucleotide conjugated to two, six, orten FITC fluorophore moieties (FITC-2, FITC-6, and FITC-10,respectively).

In separately contained reactions, three aliquots of anti-A arehybridized with aliquots of PB-2, PB-6, and PB-10, respectively,yielding the molecular probes anti-A:PB-2, anti-A:PB-6, and anti-A:PB-10(hereinafter referred to for the sake of brevity as A:2, A:6, and A:10),and three aliquots of anti-B are hybridized with aliquots of FITC-2,FITC-6, and FITC-10, respectively, yielding the molecular probesanti-B:FITC-2, anti-B:FITC-6, and anti-B:FITC-10 (hereinafter referredto as B:2, B:6, and B:10).

Nine identical aliquots of a sample of the cells of interest are labeledwith each of the nine possible two-way combinations of the molecularprobes (A:2+B:2, A:2+B:6, . . . , A:10:B10), and the nine labeled cellaliquots are then separated evaluated via flow cytometry to determinethe one two-way combination of molecular probes which optimally detectsand quantifies the abundances of Target A and Target B in the sample.

Subsequent to this assay development study, the optimal two-waycombination of the two molecular probes thus identified is employed inassays to determine the presence and abundance of Targets A and B insamples comprising this cell type.

Example 22

Prophetic Example of Labeled Antibody Catalog Simplification:

A vendor of primary-labeled antibodies whose catalog includes 800antibodies and 30 fluorophores wishes to provide customers with thewidest possible choice of antibody-fluorophore combinations without thenecessity of manufacturing and marketing 800×30=24,000 differentproducts. The vendor therefore manufactures and markets each of its 800antibodies as conjugates with a first oligonucleotide, and each of its30 fluorophores as conjugates with a second, complementaryoligonucleotide, for a total of 830 products. A customer may purchaseany antibody-oligo product in the vendor's catalog, anyfluorophore-oligo product in the vendor's catalog, and assemble them viahybridization into any arbitrary fluorophore:antibody combination via asimple hybridization reaction.

Example 23

In standard solid phase oligonucleotide synthesis the purity of thecrude oligonucleotide, either deoxy, RNA or modified backbone-basedoligonucleotides, may be approximately 80-90% depending in part on thelength of the oligonucleotide as the yield of each step approximately98-99%. The amount of failure sequences may increase as the length ofthe oligonucleotide increases. Chromatographic purification of theoligonucleotide, by either reverse phase (RP) or ion exchange (IEX), isused to increase the purity of the oligonucleotide however due to theirpoor resolution only marginally increase the purity at significant lossof yield. The yield of the oligonucleotide following chromatographicpurification is typically 30-70% and methods are laborious and requireexpensive chromatographic equipment. There is a need to prepareoligonucleotides of high purity inexpensively and in high yields withoutthe use of chromatographic equipment.

In most conjugation reaction it would be advantageous to have a linkableoligonucleotide that is of 90% or greater purity so that a higherproportion of oligonucleotide conjugates to the biomolecule or surfacebeing modified resulting in a product that contains less unconjugatedoligonucleotide. This would allow easier purification of the product orin some assays where unconjugated oligonucleotide does not substantiallyinterfere with the assay a crude product can be used directly. Certainembodiments disclosed herein are directed to the situation where thelinkable group incorporated on the oligonucleotide is an aromaticaldehyde derivative, 4-formylbenzaldehyde (4FB). FIG. 69 presents anexemplary synthetic scheme that may be used to prepare 4FB-modifiedoligonucleotides.

In this example, we demonstrate methods in which 4FB-oligonucleotidescan be purified by immobilization on hydrazide-beads, washing away thefailure sequences and releasing the 4FB-oligonucleotide from thehydrazide-beads using acidic buffer containing, sulfo-benzaldehyde andaniline (see FIG. 70). The highly purified 5′-4FB oligonucleotide isprepared by (1) immobilizing the 5′-4FB-oligonucleotide on hydrazidebeads using acidic buffer, pH 5-6, including 25 to 100 mM, for example50 mM aniline, (2) wash the beads to remove failure sequences, (3)release the 5′-4-FB-oligonucleotide from the bead using a solution ofsulfo-benzaldehdye and aniline in buffer, pH 6.0 and (4) exchanging theoligonucleotide into buffer of choice by diafiltration, dialysis orother methods known to those skilled in the art. In certainapplications, the aniline may be replaced by other acceptableamino-benzene derivatives. The overall purity of the oligonucleotideisolated by this process is equal to or greater than 93% and the yieldof highly purified oligonucleotide is approximately 70-80% based ontotal oligonucleotide loaded on the beads. This method is generallyapplicable to aromatic aldehydes-modified oligonucleotides or otheraromatic aldehyde-modified biomolecules, surfaces, polymers, moleculesor combinations thereof.

Example 23 illustrates procedures using purified 4FB-oligonucleotidesthat result in 90% or greater conjugation of input oligonucleotide onHyNic-modified antibodies. FIGS. 71A (purified 4FB-oligo) and 71B (crude4FB-oligo) presents results demonstrating the efficiency of theconjugation of 4FB-oligonucleotides to a HyNic-modified antibody andFIG. 72 presents results of linking and a 4FB-oligonucleotide to aHyNic-modified peptide.

4FB-Oligonucleotide Purification:

Oligo 5′-4FB-CLINK20-A (61 mg; MW 6443; sequence5′-4FB-GGAAGCGGTGCTATCCATCT) was dissolved in Conjugation Buffer (100 mMphosphate, 150 mM NaCl, pH 6.0; 3 mL; final concentration 0.491 OD/uL).Carbolink Beads (1 mL; ThermoPierce, Rockford, Ill.) were prewashed withConjugation Buffer (3×5 mL). To the washed Carbolink beads was added5′-4FB-CLINK20-A (200 ODs) and the mixture was placed in a 40° C. waterbath for 2 h with shaking every 30 minutes then allowed to standovernight at room temperature. The beads were washed with ConjugationBuffer (5×5 mL; the combined washes were retained) The immobilizedoligonucleotide was dissociated from the bead by three treatments with asolution of 50 mM sulfo-benzaldehyde and 120 mM aniline in ConjugationBuffer (×mL) and incubation at 40° C. for 2 h and a final incubationovernight at room temperature. The combined washings were concentratedusing a 3K MWCO Vivaspin diafiltration device (Sartorius Stedim) andwashed with water. Table 7 presents the yields for the recovered unboundand bound and released oligonucleotides.

TABLE 7 % ODs 4FB-MSR recovered Initial 200.0 0.85 Unbound recovered18.1 0.05 8.1 Bound and released 157.8 1.03 79.0 recovered

Conjugation of Purified 4FB-Oligonucleotide to HyNic-Modified IgG:

IgG (100 ug at 1 mg/mL) was buffered exchanged into Modification Buffer(100 mM phosphate; 150 mM NaCl, pH 8.0) using a 0.5 mL 40K MWCO ZebaSpin Desalting Column (ThermoPierce, Rockland, Ill.). The recoveredprotein concentration was confirmed by determining its A280 absorbanceusing a NanoDrop 1100 spectrophotometer. To the antibody solution wasadded S-HyNic (35 mol equivalents) in DMF (xx uL) and incubated at roomtemperature for 3 h. The HyNic-modified IgG was desalted intoConjugation Buffer (100 mM phosphate, 150 mM NaCl, pH 5.0) containing 25mM aniline using a 0.5 mL 40K MWCO Zeba column pre-equilibrated with theConjugation Buffer/aniline buffer. To the HyNic-IgG was added purified4-FB-CLINK20-A oligonucleotide in x uL water and incubated at roomtemperature for 3 h. The product was desalted into PBS, pH 7.2containing 1 mM EDTA and 0.05% azide using a 0.5 mL 40K MWCO Zebacolumn. Table 8 presents the results of three modifications with threedifferent antibodies. The protein concentration was determined byBradford protein assay. The MSR and the number of oligos/antibody weredetermined by AUC of analytical SEC chromatogram (see, for example,FIGS. 71A and 71B) using SuperDex200 column (GE Healthcare, Piscataway,N.J.).

TABLE 8 Conc Amount Free Oligo Conjugated Conc Yield Antibody (mg/mL)(ug) (%) (%) MSR (mg/mL) (%) Herceptin 1.0 100 4.0 96.0 3.84 0.89 89 CD41.0 100 6.9 91.2 3.65 0.80 80 CD8 1.0 100 7.0 93.0 3.72 0.87 87

FIG. 69 illustrates a scheme for the incorporation of 4FB-moiety using a4FB-phosphoamidite (A) on the 5′-end of an oligonucleotide during itssolid phase synthesis. FIG. 70 illustrates a schematic representation ofthe process used to purify 4FB-oligonucleotides as described in Example23. Results are shown in FIG. 71A for conjugation of a 4FB-20meroligonucleotide (4 mol equiv) to a HyNic-modified antibody (FIG. 71A)and in FIG. 71B for a crude 4FB-20mer oligonucleotide (5 mol equiv) to aHyNic-modified antibody using the method described herein.

Conjugation of Purified 4FB-Oligonucleotide to HyNic-Peptide:

To two separate tubes containing 20 mer 5′-5-4FB-GGA AGC GGT GCT ATC CATCT-3′ (100 ug; 3.01 OD; 0.15 OD/uL) in Conjugation Buffer (20.04 uL; 100mM phosphate, 150 mM NaCl. pH 6.0) was added HyNic-Peg2-c-Myc peptide(Solulink Biosciences, San Diego, Calif.) 1.5 and 3.0 mol equivalents at4 mg/mL in water respectively followed by the addition of 1/10 volume ofTurboLink Buffer (100 mM aniline, 100 mM phosphate, 150 mM NaCl, pH 6.0;Solulink Biosciences). The reactions were incubated at room temperaturefor 2 h then 48 h at 4° C. The reactions were analyzed by PAGE and theresults are shown in FIG. 72. FIG. 72 shows a PAGE gel of theconjugation of a 20mer 4FB-oligonucleotide purified (Lane 2) to aHyNic-Peg2-9mer peptide (1.5 mol equiv (Lane 3) and 3.0 mol equiv (Lane4)). This gel was developed using Sybr gold.

Example 24

Amino-oligonucleotide-HyNic Modification:

FIG. 76 illustrates an exemplary step wise protocol that may be used tocapture and detect an antigen from a biological sample, according tocertain embodiments, wherein an antibody oligonucleotide conjugate ispreassembled on a bead immobilized with its complementary oligo. Theamino-oligonucleotide was desalted into SE Modification Buffer (100 mMPhosphate, 150 mM NaCl, 100 mM Sodium Sulfate, 10 mM EDTA, pH 7.4) usinga 3 KD MWCO VivaSpin column (4×), the concentration was adjusted between0.2-0.5 OD/ul. To the volume of amino-oligonucleotide was added a ½volume of DMF followed by addition of S-HyNic (25 equivalents in DMF).The reaction was incubated at room temperature for 2 hours, diluted to400 ul with SE Conjugation Buffer (100 mM Phosphate, 150 mM NaCl, 100 mMSodium Sulfate, 10 mM EDTA, pH 6.0) and desalted using 3 KD MWCOVivaSpin column (4×). The MSR of HyNic-modified oligonucleotide wasdetermined by mixing HyNic-oligonucleotide with 2-sulfo-benzaldhyde(SBA) reagent (0.5 mM 2-SBA in 0.1M MES buffer) and incubating at 40° C.for 1 hour, hydrazone formed between HyNic and 2-SBA forms achromophoric hydrazone that absorbs at 350 nm with a molar extinctioncoefficient of 24500, and 260 nm (oligonucleotide) on aSpectrophotometer (NanoDrop, ND-1000) are measured and MSR calculatedare shown in Table 1 (MSR of HyNic modified oligonucleotides). The OD/ulof the purified HyNic-oligonucleotide is measured and used directly inthe following conjugation reaction.

TABLE 9 MSR Name (M1/M2) HyNic-HyLk1′ 0.92 HyNic-HyLk2′ 0.73HyNic-HyLk3′ 0.75

4FB-Compel beads preparation: Compel beads (Bangs Laboratories Inc., 6.3um) (100 mg in 2.04 ml) were washed with 8 ml MES Activation Buffer(0.1M MES, 0.5 M NaCl, pH 6.0) (2×) using a magnetic rack. 41.39 mg ofEDC (Quanta Bio, Cat #10025, lot #BV34012) was weighed and dissolved in300 ul HyClone H2O. 43.98 mg of Sulfo-NHS is dissolved into 1.5 ml of1×MES Buffer (0.1M MES, pH 5.0). To Compel beads was added EDC andSulfo-NHS solutions prepared as above and reaction incubated at roomtemperature for about 20 minutes on a rotator followed by washing with10 ml of MES Activation Buffer (4×). To the washed beads was added 9 mlof 0.5 Methylenediamine in Borate Buffer (0.1M Borate Buffer, pH 8.0)and the reaction was incubated at room temperature for about 2 hours onthe rotator followed by washing with 10 ml of Modification Buffer (100mM Phosphate, 150 mM NaCl, pH 7.4) containing 0.05% Tween-20 (3×),Hyclone H2O 2O (3×), and Modification Buffer only (3×). To the washedbeads was added Sulfo-4FB solution (53.37 mg in 944.3 ul ModificationBuffer), the reaction was mixed by vortex and incubated at roomtemperature for about 2 hours on rotator followed by washing with 10 mlConjugation Buffer (100 mM Phosphate, 150 mM NaCl, pH 6.0)/0.05%Tween-20 (3×), then Conjugation Buffer alone (6×), the concentration ofthe beads was subsequently determined. Serial dilutions were preparedfrom native Compel beads at the following concentrations: 1250, 625,312, 156 and 78 ug/ml in Hyclone H2O, 200 ul of each solution was addedto each well, standards in single well and sample in duplicate, OD at600 nm was recorded and concentration of beads determined according tostandards using SOFTmaxpro software (6.8 mg/ml). To measure the4FB-binding capacity of modified beads, 44.1 uM 4-hydrazino-stibazole(SigmaAldrich) solution was prepared in 0.1M MES Buffer/0.25 M NaCl and100 ul was added to 100 ug of 4FB-Compel beads and native Compel beads,the reactions were incubated at room temperature for about one hour on ashaker and supernatant collected following centrifugation at 14,000 gfor 4 minutes. 4-Hydrazino-stibazole binds to 4FB on beads to form ahydrazone (absorbance 350 nm, molar extinction coefficient 28500). Basedon the reduction of 4-hydrazino-stibazole in the supernatant,4FB-binding capacity of Compel beads was calculated to be 7.2 nmole/mg.These 4FB-Compel beads were used in the following experiments.

HyNic-oligonucleotide/4FB-compel beads conjugation: To the 3 mg4FB-Compel beads was added HyNic-oligonucleotide (5.725 nmol/mg beads)in SE Conjugation Buffer followed by the addition of 1/10 of volume ofTurboLink Buffer (100 mM aniline, 100 mm phosphate, 150 mM NaCl, pH6.0). The reactions were incubated for 16 hours on a shaker.Unconjugated oligonucleotides were removed from Compel beads by washingwith PBS-T (10 mM phosphate, 150 mM NaCl, pH 7.4)/0.05% Tween-20) (3×)and PBS (1×) and re-suspended into PBS at 3 mg/ml. The immobilizedoligonucleotides on beads were quantified as following:

General procedure to quantify the amount of oligonucleotide immobilizedon beads: The following example is representative of the protocol used.

Step 1) Complementary oligonucleotide/Dy490 modification. Amino-HyLk1was desalted (4×) using a 3 KD MWCO VivaSpin column into ModificationBuffer followed by adjusting the concentration to 0.5-0.6 OD/ul. ToAmino-HyLk1 (10.5 OD in 18.6 ul; 0.56 OD/ul) was added a solution ofDy490 in anhydrous DMF (0.75 mg in 10 ul; 15 mol equiv) as shown inTable 10. The reaction mixture was incubated at room temperature forabout 3 hours then desalted into PBS buffer using a 3 KD MWCO column(9×) until the flow through was clear of fluorescence. The % Dy490incorporation on the oligonucleotides was determined by OD readings at490 nm and 260 nm on a Spectrophotomemter (NanoDrop, ND-1000), as a peakis formed by modified Dy490 at 490 nm (OD 260 nm correction factor0.235). The MSR of Dy490-modified oligonucleotides are listed in Table10 (Oligonucleotides modified with Dy490). The OD/ul is determined andadjusted to 0.2 OD/ul and used in the following hybridization procedure.

TABLE 10 Oligo MSR HyLk1 1.06 HyLk2 1.03 HyLk3 0.98

Step 2) Hybridization of Compel beads-HyLkX′ with Dy490-HyLkX: 50 ugCompel-HyLkX′ beads were washed with PBS-T (1×) then PBS (1×),centrifuged at 5000 g for 5 minutes to remove the supernatant. A 400 nMDy490-HyLkX solution was prepared in PBS. To the Compel-HyLkX′ beads theDy490-HyLkX solution was added. The Dy490-HyLkX solution in the absenceof beads or that received 4FB-compel beads without oligonucleotideconjugated were included as controls. The hybridization reaction wasincubated at room temperature for 30-60 minutes on a shaker. Followingcentrifugation at 5000 g for 5 minutes, supernatant was collected formeasuring fluorescence in solution and beads were washed with PBS/0.05%Tween-20 (2×) and PBS (1×) and subjected to FACS acquisition.

Step 3) Creation of a Dy490 standard curve using Dy490 NHS Ester: A 0.5mg/ml solution of Dy490 in DMF was prepared. A 4000 nM Dy490 NHS Estersolution was prepared in PBS and 1:1 serial dilutions were prepared atthe following concentrations: 4000, 2000, 1000, 500, 250, 125 and 62.5nM. Each concentration was read on a fluorescence spectrophotometer(NanoDrop 3300) five times to create a linear standard curve for Dy490fluorescence.

Step 4) The amount of HyLkX-Dy490 hybridized to the beads was determinedusing the following procedure: The fluorescence of the supernatant wasmeasured and subtraction of concentration of each sample from theconcentration of Dy490-HyLkX solution in the absence of beads to obtainthe reduction of Dy490-HyLkX in each sample, yielded the amount ofoligonucleotides being hybridized onto the beads, based on the fact that50 ug beads in each sample are tested, immobilized oligonucleotides onbeads in nmol/mg were calculated and listed in Table 11(Oligonucleotides immobilized on Compel beads).

TABLE 11 Compels Immobilized oligonucleotides Name Beads (mg) (nmol/mg)HyLk1′ 3 1.09 HyLk2′ 3 1.02 HyLk3′ 3 0.79

Step 5) Quantification of immobilized oligonucleotides on beads by FACS.Hybridized Compel beads were subjected to FACS acquisition after washingand 20,000 events collected under an appropriate setting. Results wereanalyzed as peak appeared on FL1 and “mean fluorescence intensity” (MFI)of each beads sample compared.

Protocol for preparation of a protein/oligonucleotide.

Step 1) BSA-HyNic modification: BSA (Jackson Immuno Research) solutionwas prepared at 10 mg/ml initial concentration and desalted tomodification buffer using a 5 ml 7 KD MWCO Zeba column, theconcentration was adjusted to 5 mg/ml after desalting. To the solutionof BSA (3.6 ml of a 5 mg/ml solution) was added a solution of S-HyNic(71.4 ul of a 22.6 mg/ml solution in anhydrous DMF; 20 mol equiv). Thereaction was gently vortexed and then incubated at room temperature forabout 3 hours. The reaction mixture was desalted into conjugation bufferusing a 10 ml 40 KD MWCO Zeba column and the concentration of BSAdetermined by BCA assay. The MSR of BSA-HyNic was 10.4.

Step 2) BSA-HyLkX conjugation using BSA-HyLk1 as the example: ToHyNic-BSA (1.6 ml of 3.37 mg/ml in conjugation buffer) prepared in Step1 was added 4FB-HyLk1 (Trilink, 4FB MSR 0.51) (158 ul of 0.642 OD/ul inconjugation buffer; 3 functional mole equivalent) and a 1/10 of volumeof TurboLink Buffer (100 mM aniline, 100 mM phosphate, 150 mM NaCl, pH6.0) was added. The reaction was mixed and incubated at room temperaturefor about 2 hours then 4° C. for about 16 hours. BSA-HyLk 1 conjugatewas purified by size exclusion chromatography (Superdex200; GEHealthCare) using PBS as eluant at 0.5 ml/min (75 min run) and theconjugate product collected between 15-26 min. The MSR of BSA-HyLk Xwere determined by area under the curve of the chromatograms and shownin Table 12 (MSR (oligos/BSA) of BSA-oligonucleotide conjugated).

TABLE 12 Conjugate MSR BSA-HyLk1 2.96 BSA-HyLk2 2.95 BSA-HyLk3 3.00

Biotin/anti-rabbit IgG modification: Anti-rabbit IgG (JacksonImmunoResearch) was desalted into Modification Buffer using a 2 ml 7 KDMWCO Zeba Column and concentration determined (2.15 mg/ml) by NanoDrop(E1%=13.6). A solution of Sulfo-ChromaLink Biotin (Solulink) (18.4mg/ml) in anhydrous DMF was prepared. To anti-rabbit IgG (0.84 mg; 391ul of a 2.15 mg/ml solution) was added Sulfo-ChromaLink Biotin (4.2 ul;15 mol equiv). The reaction mixture was incubated at room temperaturefor about 3 hours then desalted into PBS using a 2 ml 40 KDMWCO ZebaColumn and the concentration determined to be 1.35 mg/ml by BCA assayand biotin incorporation of 5.34 biotins/antibody was determinedspectrophotometrically using chromophoric bis-arylhydrazone linker ofChromaLink Biotin (A354, extinction coefficient 29,000).

Flow cytometric analysis of capturing antibody (anti-BSA) in solution byantigen (BSA) hybridized on beads through oligonucleotide and detectedby biotin-2^(nd) antibody followed by Streptavidin-RPE: HyLk1′-compelbeads (50 ug/test; 17 ul of 3 mg/ml) were washed with PBS/T (PBS/0.05%Tween-20) followed by adding a 50 ul of PBS solution containingHyLk1-BSA conjugate (644 ng; 23 ul of 2.8 mg/ml). The hybridizationreaction was incubated on a shaker at room temperature for about 45minutes. The beads were washed with PBS/T (3×) by centrifugation at 5000g for 5 minutes each time. Serial dilutions of anti-BSA (Fitzgerald)were prepared as the following: 366, 36.6, 3.66, 0.366, 0.0366 ng into50 ul of PBS from 1 mg/ml solution and into which 50 ug beadsre-suspended. The beads were incubated on the shaker at room temperaturefor about 1 hour followed by PBS/T wash (3×). To beads 50 ul of biotinmodified anti-rabbit IgG (0.2 ug; 50 ul of 4 ug/ml) solution in PBS wasadded and incubated on the shaker at room temperature for about one hourfollowed by PBS/T wash (3×). To beads 50 ul ofStreptavidin-R-Phycoerythrin (SAPE) (Invitrogen) (0.03 ug; 50 ul of 0.68ug/ml) solution in PBS was prepared and added and the beads wereincubated on the shaker at room temperature for around 30 minutesfollowed by PBS/T wash (3×). The beads were finally re-suspended into300 ul PBS and subjected to acquisition on FACSCalibur, 20,000 eventswere collected per sample. Debris and clumps of beads were excluded bygating based on FSC vs. SSC dot plots. Analysis of PE+ beads populationwas performed using FlowJo software (Tree Star, Inc). Flow results arepresented in FIG. 75.

Example 25

Preparation of Antibody-oligonucleotides conjugates of increasingoligonucleotide/antibody ratios using size exclusion chromatograph.

To α-CD4 antibody (3.42 mg at 4.94 mg/mL in Modification Buffer) wasadded S-HyNic (300 uL of 20 mg/mL solution in anhydrous DMF; 21 molequiv). The reaction mixture was incubated at room temperature for 3hours and desalted into Conjugation Buffer using 2×2 mL 7K MWCO Zebacolumns. The protein concentration (BCA assay) was determined to be 3.83mg/mL and the HyNic substitution ratio was 7.3 HyNic/α-CD4. TheHyNic-modified α-CD4 antibody was divided into five tubes eachcontaining 0.63 mg (165 uL). A solution of a 20mer5′-4FB-oligonucleotide (MW 6312; 0.463 OD/uL) in nuclease free water wasprepared. To the five aliquots of HyNic-α-CD4 were added 2.27, 3.98,5.68, 7.95, 11.36 and 15.0 mol equiv of 5′-4FB-oligonucleotide followedby the addition of 1/10 volume of TurboLink Buffer (100 mL phosphate,150 mM NaCl, 100 mM aniline, pH 6.0; Solulink Biosciences, San Diego,Calif.). The conjugates were purified by size exclusion chromatographyusing a Superdex200 column (GE Healthcare). Table 13 presents the datacharacterizing the α-CD4-oligonucleotide conjugate products.

TABLE 13 Equiv 4FB- Equiv 4FB-oligo oligo added incorporated mg/mL 2.271.92 0.113 3.98 3.34 0.112 5.68 4.64 0.113 7.95 5.52 0.107 11.36 6.490.123 15.00 7.87 0.109

Flow cytometric analyses of α-CD-oligonucleotide conjugates ofincreasing oligonucleotide/antibody ratios: The conjugates prepared asdiscussed herein were hybridized to their complementaryoligonucleotide/dextran/Dy490 detector in 1/1 mole ratio based onincorporated oligonucleotides for 15 min at room temperature, added tocells for 30 min at 4° C., washed twice with PB. The conjugates wereadded to normal B6 mouse splenocytes as in Example 14 and analyzed on aFACS Canto using a 488 laser with a FITC filter. Results are presentedin FIG. 73. FIG. 73 shows the flow cytometric results analyzingantibody-oligonucleotide conjugates hybridized to its complementaryoligonucleotide/dextran/poly-Dy490 detector with respect to the numberof oligosnucleotides conjucated to the antibody. Here an α-CD4-antibodyconjugated to increasing ratios of oligonucleotide/antibody hybridizedto its detector complement were incubated with B6 mouse cells andanalyzed by flow cytometry as described in Example 14.

Example 26

Prophetic Example Imaging:

A researcher wishing to determine the percentage of cells bearing amolecular target in a fixed, adherent cultured cell sample in a dishlabels the sample first with a DNA-binding first fluorophore to labelcell nuclei, then washes the sample to remove unbound DNA-binding dye,then labels the sample with a molecular probe comprising an antibodyrecognizing the target of interest conjugated to a first oligonucleotidehybridized to a second, complementary oligonucleotide conjugated to asecond fluorophore using certain embodiments disclosed herein. Afterwashing the sample to remove unbound molecular probe the sample isimaged in a fluorescence microscope and two fluorescent images areelectronically recorded—one at the emission wavelength of the firstfluorophore (labeling cellular nuclei), and one at the emissionwavelength of the second fluorophore (labeling cells positive for themolecular target). Employing conventional image analysis algorithms thetotal number of cells in the field is counted, the number of cellspositive for the molecular target is counted, and the percentage ofcells bearing the molecular target is calculated.

Example 27

Prophetic Example Imaging:

A histologist wishing to detect cells positive for a molecular target ina sectioned tissue specimen labels the tissue section with a molecularprobe comprising an antibody recognizing the target of interestconjugated to a first oligonucleotide hybridized to a second,complementary oligonucleotide conjugated to either a fluorophore such asFITC or a chromophore-generating enzyme such as horseradish peroxidaseusing certain embodiments disclosed herein. The tissue section is nextcounter-stained with hematoxylin. The labeled tissue specimen is thenobserved microscopically (employing transmitted light microscopy in thecase of a chromophore-labeled sample or fluorescence microscopy in thecase of a fluorophore-labeled sample) and is manually and/orautomatically scored for the presence/absence, abundance, distributionof label or combinations thereof.

Example 28

Prophetic Example Strip and Re-Probe:

A researcher desiring to identify a sub-population of cells in achemically fixed adherent cell sample which contains six targets, TargetA through Target F, uses a fluorescence microscope with only threeavailable optical channels, Channels A, B, and C. It is therefore notpossible for the researcher to measure the six targets simultaneously,or substantially simultaneously, using antibodies labeled with sixdifferent-colored fluorophores. The researcher therefore adopts thefollowing ‘strip-and-reprobe’ experimental strategy.

The researcher first labels the cell sample with a cocktail comprisingantibodies A, B, and C (Ab-A, Ab-B, and Ab-C, recognizing Targets A, B,and C, respectively) conjugated to oligonucleotides 1, 2 and 3,respectively, and hybridized with complementary oligonucleotides 1′, 2′,and 3′ respectively, conjugated to fluorophores X, Y, and Z respectivelyusing certain disclosed embodiments. The researcher then images thelabeled adherent cell sample and records the locations on the dish ofthe cells positive for the Targets A, B, and C simultaneously orsubstantially simultaneously.

Next, the researcher strips the fluorescent signal moieties from themolecular probes bound to the cells by subjecting the sample toconditions of temperature and ionic conditions sufficient to dehybridizethe molecular probes' oligonucleotide pairs, and washes away thedehybridized signal moieties using certain disclosed embodiments. Aftervisually confirming the completeness of stripping (observing no residualfluorescent signals), the researcher next re-probes the cell sample(under temperature and ionic conditions sufficient to maintainoligonucleotide hybridization) by labeling it with a cocktail comprisingantibodies D, E, and F, recognizing Targets D, E, and F, respectively,conjugated to oligonucleotides 1, 2 and 3, respectively, and hybridizedwith complementary oligonucleotides 1′, 2′, and 3′ respectively,conjugated to fluorophores X, Y, and Z respectively using certaindisclosed embodiments. The researcher then images the labeled adherentcell sample (observing the same fields observed in the first round ofstaining) and records the locations on the dish of the cells positivefor the Targets D, E, and F simultaneously or substantiallysimultaneously. Cells marked in the first round of labeling assimultaneously, or substantially simultaneously, positive for Targets A,B, and C and marked in the second round of labeling as simultaneously,or substantially simultaneously, positive for Targets D, E, and F aretherefore simultaneously, or substantially simultaneously, positive forTargets A, B, C, D, E and F. thereof.

Example 29

Prophetic Example Panel Optimization:

In the development of a flow cytometry assay it is desired to detect,distinguish, quantify or combinations thereof three separatesub-populations of cells in a sample, employing a reagent cocktail ofthree differently fluorescently labeled antibodies, Ab-A, Ab-B, andAb-C, against three targets, Target A, Target B and Target C,respectively. After ruling out several possible fluorophores forpractical considerations, the assay developer may, in principle, choosefrom among four remaining fluorophores (Fl-1 through Fl-4) to label thethree antibodies, but faces a multitude of issues impacting the bestchoice of three from among these four, as well as the best choice ofwhich fluor to place on which antibody, including at least one or moreof the following considerations regarding spillover of signal betweendetection channels, different degrees of non-specific binding of thefluorophores to the cells, differential sensitivities of thefluorophores to photobleaching, and the differing intrinsic brightness'sof the fluors due to their different quantum yields.

A collection of separately contained reagents is provided, comprisingthree tubes of Ab-A, Ab-B, and Ab-C, respectively, each conjugated to afirst oligonucleotide, and four tubes of Fl-1 through Fl-4,respectively, each conjugated to a second oligonucleotide complementaryto the first oligonucleotide using certain disclosed embodiments. In 12separately contained reactions each antibody is hybridized to eachfluorophore, respectively, yielding the molecular probes Ab-A:Fl-1,Ab-A:Fl-2, . . . , Ab-C:Fl-4 using certain disclosed embodiments. Intoseparate tubes these molecular probes are next combined in the 24possible 3-way combination cocktails (Ab-A:Fl-1+Ab-B:Fl-2+Ab-C:Fl-3,Ab-A:Fl-1+Ab-B:Fl-2+Ab-C:Fl-4, . . . , Ab-A:Fl-4+Ab-B:Fl-3+Ab-C:Fl-2).

Twenty-four identical, or substantially identical, aliquots of a sampleof the cells of interest are labeled with each of the 24 cocktails,respectively, and the 24 labeled cell aliquots are then separatedevaluated via flow cytometry to identify the one cocktail compositionwhich optimally detects, differentiates, and quantifies the sample'sthree sub-populations.

Subsequent to this assay development study, the optimal three-componentcocktail thus identified is employed in assays to determine the presenceand abundance of the three sub-populations of cells in samples usingcertain disclosed embodiments.

Example 30

Immunoturbidity:

4FB-OptiLink bead preparation: Carboxy-modified OptiLink beads assupplied (750 mg; 7.5 mL; Thermo Scientific Cat #83000550100290, 2.07 umin diameter) were dialyzed into 100 mM MES buffer pH 6.0 by injectingthe suspension of beads into a Pierce Slide-A-Lyzer dialysis cassette(3.5 kD MWCO) and placing the cassette in 2 L 100 mM MES buffer pH 6.0with gentle magnetic stirring for 6-16 hours (3×). The bead suspensionwas transferred to a 50 mL conical tube with a syringe. 819 mg of EDC(Quanta Bio, Cat #10025) was dissolved in 4 mL HyClone molecular biologygrade water (cat #SH30538.03). 854 mg Sulfo-NHS (Solulink cat #S-4020)was dissolved in 15 mL MES buffer. Both of these solutions were added tothe OptiLink beads, which were incubated at room temperature for 30minutes on a rotator. The bead suspension was dialyzed again asdescribed above and then transferred to 50 mL conical vials. A solutionof ethylenediamine in Borate Buffer (0.5 M in 0.1M borate, pH 8.0) wasadded to the beads and the reaction was incubated at room temperaturefor 12 hours on the rotator. The bead suspension was then dialyzed asdescribed above into modification buffer (100 mM Phosphate, 150 mM NaCl,pH 7.4) and transferred into 50 mL conical vials. 1.453 g Sulfo-S-4FBwas weighed and dissolved in 30 mL modification buffer. This solutionwas added to the beads, which were then incubated at RT for 16 hours ona rotator. The bead suspension was then dialyzed as described above inconjugation buffer (100 mM Phosphate, 150 mM NaCl, pH 6.0).

The concentration of the beads was subsequently determined. Serialdilutions were prepared from native OptiLink beads at the followingconcentrations: 197.6, 98.8, 49.4, 24.7 and 12.35 ug/ml in PBS (10 mMphosphate, 150 mM NaCl, pH 7.4). Similarly, a 2 uL aliquot of4FB-OptiLInk beads was diluted into 998 uL PBS (in duplicate). OD at 600nm was recorded and concentration of beads determined (32.33 mg/mL)according to the standard curve generated from OD600 readings of thenative beads. To measure the 4FB-binding capacity of the modified beads,a 246 uM S-Tag HyNic peptide solution was prepared in conjugationbuffer. 100 uL of this solution was added to 0.5 mg 4FB-OptiLink beads(in duplicates) and 0.5 mg native OptiLink beads (in duplicates). Thereactions were incubated at room temperature for 1 h on a shaker andsupernatant collected following centrifugation at 10,000×g for 10minutes. Based on the reduction of S-Tag HyNic peptide supernatant (asmeasured by OD 280), the 4FB-binding capacity of the OptiLink beads werecalculated to be 28.0 nmol/mg. These 4FB-OptiLink beads are used in thefollowing experiments.

Amino-oligonucleotide-HyNic Modification: The amino-oligonucleotides(HyLk-4′ and HyLk-5′) were desalted into Modification Buffer (100 mMPhosphate, 150 mM NaCl, pH 7.4) using a 3 kD MWCO VivaSpin column andcentrifuging at 15,000×g for 10 minutes (4×). The concentration wasadjusted between 0.3-0.5 OD/uL. To the volume of amino-oligonucleotidewas added a ½ volume of DMF followed by addition of S-HyNic (20 mg/mL inDMF; 25 equivalents). The reaction was gently vortexed for 5 seconds andthen incubated at room temperature for 2 hours. The solution was thendiluted to 400 uL with Conjugation Buffer (100 mM Phosphate, 150 mMNaCl, pH 6.0) and desalted using 3 kD MWCO VivaSpin column bycentrifuging at 15,000×g for 10 minutes (4×). A280 measurements ofHyNic-HyLk-4′ and -5′ were measured to determine their finalconcentrations.

HyNic-oligonucleotide immobilization onto 4FB-OptiLink beads: To threealiquots of 4FB-OptiLink beads were added HyNic-oligonucleotide at 3different equivalents for each oligo (14, 1.4, and 0.14 nmol/mg beads)in Conjugation Buffer followed by the addition of 1/10 of volume ofTurboLink Buffer (100 mM aniline, 100 mm phosphate, 150 mM NaCl, pH6.0). The reactions were incubated for 16 hours on a shaker.Unconjugated oligonucleotides were removed from the beads by washingwith PBS (10 mM phosphate, 150 mM NaCl, pH 7.4) after centrifuging thebeads at 5,000×g for 10 minutes (4×). The beads were re-suspended in PBSat 10 mg/ml. Table 14 shows Oligonucleotides immobilized on OptiLinkbeads.

TABLE 14 Functional Equiv of Aniline Total reaction OptiLink beadsoligonucleotide con. volume Oligo (mg) (nmol/mg) (mM) (uL) HyLk4′ 5 14,1.4, 0.14 10 100 HyLk5′ 5 14, 1.4, 0.14 10 100

α-bIgG-HyNic modification: Rabbit Anti-Bovine IgG (H+L) (α-bIgG)(Jackson Immuno Research, cat #301-005-003) solution was prepared at 2.4mg/ml initial concentration and desalted to modification buffer using a2 mL 7 kD MWCO zeba column. To the solution of α-bIgG (443.1 ug; 210 uLof a 2.11 mg/ml solution) was added a solution of S-HyNic (3.58 uL of a4.8 mg/ml solution in anhydrous DMF; 20 mol equiv). The reaction wasgently vortexed for 5 seconds and then incubated at room temperature for3 hours. The reaction mixture was desalted into conjugation buffer usinga 2 mL 40 kD MWCO zeba column and the concentration of α-bIgG determinedby BCA assay.

α-bIgG-HyLkX conjugation (using IgG-HyLk4 as the example): ToHyNic-α-bIgG (210 ug; 210 uL of 2.00 mg/mL in conjugation buffer)prepared in Step 4 was added 4FB-HyLk4 (IDT, 4FB-MSR=0.33) (2.789 uL of0.91 OD/uL in conjugation buffer; 3 functional mole equivalents) and a1/10 of volume of TurboLink Buffer (100 mM aniline, 100 mM phosphate,150 mM NaCl, pH 6.0) was added. The reaction was mixed and incubated atroom temperature for 4 hours and then 4° C. for 16 hours. α-bIgG-HyLk4conjugate was purified by size exclusion chromatography (Superdex200; GEHealthCare) using PBS as eluent at 0.5 ml/min (45 min run) and theconjugate product collected between 11-17 min. The MSR of the conjugate(number of oligos per α-bIgG) was determined by the area under the curveof the chromatograms from the HPLC trace. α-bIgG-HyLk conjugates werethen concentrated to approximately 100 ug/mL with 10 kD MWCO Amiconfilter units (Millipore cat #UFC801024). Concentrations of theconjugates were determined by BCA assay. Table 15 shows the MSR(oligos/α-bIgG) of α-bIgG-oligo conjugate.

TABLE 15 Conjugate Functional 4-FB oligo added (equiv) MSR α-bIgG-HyLk43 2.98 α-bIgG-HyLk5 3 2.95

Preparation of control α-bIgG-OptiLink beads via EDC coupling: 9 mg (1mL of a 9 mg/mL suspension) of OptiLink amino beads was washed with 1 mL100 mM MES buffer, pH 5.0 (3×). Beads were vortexed for 20 seconds andsonicated for 20 seconds during each wash; beads were centrifuged at10,000×g for 10 minutes to form a pellet from which the supernatantcould be decanted. The beads were then suspended in 0.5 mL 100 mM MESbuffer pH 5.0. To this suspension, a freshly prepared solution of 10.73mg of EDC dissolved in 0.5 mL 100 mM MES buffer, pH 6.0 was added. Beadswere then spun on a rotator at RT for 20 minutes. Beads were then washedas described above 3 times, and resuspended in 0.5 mL 100 mM MES buffer,pH 6.0. 309 ug of α-bIgG (140 uL of a 2.21 mg/mL solution of α-bIgGdesalted into 100 mM MES buffer pH 5.0 with a 2 mL 7K MWCO zeba column)was then added to the beads. The beads were then spun on a rotator for 4hours at RT. Beads were then washed 3 times as previously described, andwere finally resuspended in 0.5 mL PBS (10 mM phosphate, 150 mM NaCl, pH7.4).

Bovine IgG preparation: Bovine IgG (Sigma Aldrich γ-Globulins frombovine blood cat #G7516) (BIgG) was desalted using a 7 kD MWCO zebacolumn into modification buffer. This solution was concentrated to 1mg/mL as determined by BCA assay. Dilutions were made into modificationbuffer to the following levels: 1 mg/mL, 100 ug/mL, 10 ug/mL, 1 ug/mL,100 ng/mL, 50 ng/mL, 10 ng/mL, and 5 ng/mL.

Agglutination: Samples of PBS, HyLkX′-modified beads/HyLkX-α-bIgGconjugate or EDC-coupled OptiLink-anti Bovine IgG beads were preparedand added to a 96-well plate. Serial dilutions by half were madestarting with 109.68 ug beads per well down to 0.86 ug beads per well.The plate was then placed on a shaker at RT for 45 minutes to allow forhybridization and therefore immobilization of the aBIgG to theHyLk-OptiLink beads. A volume of the appropriate solution of α-bIgGsolution was then added to the appropriate wells. Amounts of 100 ng, 10ng, and 1 ng of α-bIgG were added to each amount of beads. The degree ofagglutination was monitored both visually and by OD600. Results of thetest are summarized in table 16 and are shown in FIG. 74. Table 16provides the results of agglutination test with HyLk5′-OptiLink beadsand the results shown in Table 16 indicate that the same level ofantigen can be detected with 16 times fewer HybriLink-coupled beads whencompared to the traditional EDC-coupled beads.

TABLE 16 EDC Beads HybriLink Beads 0 ng 10 ng 1 ng 0 ng 100 ng 10 ng 1ng Ag Ag Ag Ag Ag Ag Ag 1 2 3 4 5 6 7 8 9 10 11 12 A − + + + +− + + + + + + B − + + + + − + + + + + + C − − − − − − + + + + + + D − −− − − − + + + + + + E − − − − − − + + + + + + F − − − − − − + + + + + +G − − − − − − − − − − − − H − − − − − − − − − − − −

Example 31

This example is directed to a method for detecting one or morebiological targets of a complex sample in a detection assay usingcomplementary detectors to which the fluor or signal generation moietyare directly conjugated. A-CD4 and α-CD8a were conjugated to the 20, 30and 40mer amino-oligonucleotides listed in the Table 17 using theprotocol described in Example 1. Table 17 also lists the MSR(oligonucleotides/antibody).

TABLE 17 Antibody Sequences MSR α-CD4 (Clone GK1.5)(20 mer) 5'-C6-amino-GGA AGC GGT GCT ATC CAT CT 3.0(30 mer) 5'- C6-amino-CAC CCA GCC GAT GAC CTC TTA 2.9 GTT TCA CGC -3(40 mer) 5'- C6-amino-CAC CCA GCC GAT GAC CTC TTA 3.1GTT TCA CGC AAA GCA CAC G -3' α -CD8a (Clone 53-6.7)(20 mer) 5'- C6-amino-GGA AGC GGT GCT ATC CAT CT 2.8(30 mer) 5'- C6-amino-CAC CCA GCC GAT GAC CTC TTA 3.0 GTT TCA CGC -3(40 mer) 5'- C6-amino-CAC CCA GCC GAT GAC CTC TTA 3.0GTT TCA CGC AAA GCA CAC G -3'

Fluorophore labeling of complementary amino-modified oligonucleotides:The complementary 20, 30 and 40mer multi-amino-modified oligonucleotides(Table 18) were conjugated to Dy490 and Dy649 dyes using protocolsdescribed in Example 12-A; Step 3B. The amino-modified thymidines in thesequences are denoted by (T*) in the Table 18. The MSR(dyes/oligonucleotide) are also listed in the Table 18.

TABLE 14 Complementary Multi-fluor Sequences Fluor(s) SR(20 mer) 5′-C6-amino-AGA TGG A(T*)A GCA CCG CTT CC- C3-amino -3′ Dy490.1 (30 mer) 5′-C6-amino- Dy490 .6GCGTGAAAC(T*)AAGAGGTCA(T*)CGGCTGGGTG-C6-amino -3′ Dy649 .6(40 mer) 5′- (C6-NH2)- Dy490 .5CGTGTGCTT(T*)GCGTGAAAC(T*)AAGAGGTCA(T*)CGGCTGGGTG-C6-amino) -3′ Dy649 .4

Bovine Flow cytometry results: The antibody-oligonucleotide/multi-fluorcomplementary oligonucleotide conjugate hybrids were prepared byincubating the antibody-oligonucleotide (1 mol equiv) with multi-fluorcomplementary oligonucleotide conjugate (4 mol equiv) for about 1 h atroom temperature. The hybrids (500 ng) were added to splenocytes(1,000,000) and incubated at 4° C. for about 30 min, washed and analyzedby flow cytometry. FIG. 79 presents the flow cytometry results of thebinding of α-CD8 20, 30 and 40mer hybrids to CD8⁺ splenocytes.

Example 32

Immunomagnetic Depletion Procedure:

Step 1) Preparation of Oligonucleotide and Antibody Conjugates.Biotinylated HyLk1′ oligonucleotides were commercially synthesized (IDT,Coralville, Iowa). Oligonucleotides were biotinylated at the 5′ end,with either no “spacer” sequence between the biotin group and theHybriLink sequence (5′-BIO:GTACTTCCTTAAACGACGCAGG-3′), or having a44-base repeating TTAA spacer inserted between the biotin modified endand the HyLk1′ sequence(5′-BIO:TTAATTAATTAATTAATTAATTTTAATTAATTAATTAATTAATTGTACTTCCTTAAACGACGCAGG-3′. Her2 antibody (Herceptin®, Genentech, California) was eitherconjugated to oligo HyLk1 as previously described (Herceptin:HyLk1), orbiotinylated (Herceptin:Biotin) and affinity purified by SoluLinkBiosciences.

Step 2) Preparation of HyLk1′ oligo- or Herceptin IgG-conjugatedmagnetic nanoparticles. Streptavidin (STV)-coated magnetic nanoparticles(0.8 μm, NanoLink™, SoluLink Biosciences) were transferred at 1 mg/mLinto wash buffer (50 mM Tris-HCl, 150 mM NaCl, with 0.05% Tween-20, pH8.0.) Washed nanospheres were separated from solution using a magneticstand. To prepare antibody labeled beads, nanospheres were blocked at 1mg/mL for 30 m at 24° C. in a blocking buffer of sterile-filteredcasein-TBS (Blocker™, Pierce Protein Research Products.) Blockednanospheres were washed 3× in TBS-T prior to the addition of antibody.Nanospheres to which oligonucleotides were conjugated were leftunblocked per manufacturer protocol. Conjugation of biotinylatedoligonucleotide was conducted as follows: Oligonucleotides weresolubilized in sterile H20 to 1 nmol/μL and combined in equal portionsof (BIO:H1′):(BIO:44:H1′). The mixed oligo solution was diluted to 10.4nmol/mL into wash buffer and immediately applied to washed nanospheresat 250 μL/mg beads (=2.6 nmol/mg). Conjugation was allowed to proceedfor 30 m at 24° C. with rotation. Conjugation of biotinylated antibody(αHer2:BIO) was conducted as follows: Protein content ofHerceptin-biotin was confirmed by BCA assay (Pierce Protein ResearchProducts). Antibody was applied at 40 μg/mL in 250 μL TBS-T/mgnanospheres. Conjugation was allowed to proceed for 30 m at 24° C. withrotation. Following conjugation, supernatants from both sets ofnanospheres (oligo- or antibody-labeled) were analyzed for remainingconjugate, either by A260 assay (oligo supernatant) or BCA assay(Herceptin supernatant). Oligo-conjugated nanospheres were determined tohave 290 pmol HyLk1′/mg solids. Antibody-conjugated nanospheres weredetermined to have 30 μg IgG/mg solids. Conjugated nanospheres werestored overnight in TBS-T at 4° C. and used for immunomagnetic depletionthe following day.

Step 3) Cell culture and tumor cell “spiking”. SKBR-3 humanadenocarcinoma cells (ATCC) were cultured in McCoy's 5A growth mediumsupplemented with 10% fetal bovine serum (FBS, Gemini BioSciences), 4 mML-glutamine (Gemini), and 1 KU/1 KU penicillin/streptomycin solution(‘pen/strep’, Invitrogen). CCRF-CEM human T-cell lymphocytes werecultured in RPMI-1640 culture medium supplemented with 10% FBS and 1KU/1 KU pen/strep. All cultures were grown in sterile, culture treatedflasks in a humidified, 37° C. incubator maintained at 5% CO2. Justprior to immunodepletion, adherent SKBR-3 cells were harvested bytrypsinization of the monolayer, followed by transfer to conical tubes.Non-adherent CCRF-CEM cells were transferred to conical tubes withouttrypsinization. Both cultures were pelleted by centrifugation for 5 minat 800×g, and resuspended in a buffer of 0.2% bovine serum albumin inphosphate buffered saline (BSA-PBS). After counting cells per mL, 10%tumor cells were added (“spiked”) into CCRF-CEM lymphocytes. Samples forimmunodepletion were aliquoted at 1×10⁶ cells/mL prior to downstreamhandling.

Step 4) Herceptin labeling of spiked cell preparations. Prior toHerceptin labeling, 1×10⁶ ‘spiked’ cells were blocked for 30 m at 24° C.in 100 μL of 0.2% BSA-PBS buffer containing 10 μL human Fc receptorantibodies (αFcX “TruStain”, BioLegend, San Diego, Calif.) Withoutwashing, 15 μg of Herceptin:HyLk1 or 15 μg of Herceptin:Biotin was addedto blocked cells. Labeling was allowed to proceed for 60 m at 4° C. withrotation. Cells were washed 2× in 0.2% BSA-PBS prior to magneticdepletion.

Step 5) Immunomagnetic depletion. Conjugated magnetic nanospheres werewashed 3× in 1 mL PBS/mg solids to remove traces of Tween-20 detergent.500 μg nanospheres were added to washed, labeled or unlabeled cells. ForHyLk1′ conjugated nanospheres, 500 μg solids at 290 nmol oligo/mg solidspresented 145 nmol HyLk1′ per sample. According to current literaturereporting 2×10⁶ Her2 molecules per SKBR-3 tumor cell×0.1×10⁶ tumor cellsper sample=2×10¹¹ Her2 molecules=333 fmol Her2 per sample. Assuming 100%antibody binding, and a molar substitution ratio of 4.0 oligos per IgGmolecule, this translates to 1.3 pmol oligo per labeled cell sample. 145nmol HyLk1′ immobilized on beads thus represents >100× excess ofavailable complementary oligo to hybridize to labeled cells. ForHerceptin:biotin conjugated nanospheres, 500 μg presents 15 μg IgG persample, the same amount of Herceptin used to label cells in theHybriLink test group. Following addition of 500 μg nanospheres persample, immunomagnetic capture (depletion) of Her2+ cells was allowed toproceed for 60 m at 4° C. with rotation. The microspheres were thenisolated from cell suspension by magnetic separation. Cellularsupernatants were placed on ice. Spheres were washed in 2×500 μL of 0.2%BSA-PBS and sample washes were combined with sample supernatants foranalysis by flow cytometry of depleted cellular preparations.

Description of sample groups. Samples included the followingpreparations: (A) Unlabeled cells without nanospheres (undepletedsample). (B) Herceptin-HyLk1 labeled cells plus unlabeled nanospheres(mock control 1). (C) Unlabeled cells plus unlabeled nanospheres (mockcontrol 2). (D) Unlabeled cells plus Herceptin:Biotin conjugatednanospheres (conventional, state-of-the-art method). (E)Herceptin:Biotin-labeled cells, plus streptavidin nanospheres(modification of conventional method). (F) Herceptin:HyLk cells plusHyLk1′ labeled nanospheres (HybriLink novel method). See FIG. 81.

Step 6) Fluorescent staining of depleted cell samples.Fluorescent-conjugated, monoclonal antibodies against Her2 (αHer2:PE,BioLegend) and tumor cell antigen EpCAM (αEpCAM:APC, BioLegend) wereapplied to cells in a staining cocktail of 125 ng αHer2:PE plus 30 ngαEpCAM:APC in 100 μL of 0.2% BSA-PBS for 60 m at 4° C. with rotation.Samples were pelleted by centrifugation for 5 m at 800×g, resuspended in250 μL 0.2% BSA-PBS, and immediately analyzed by cytometer. Undepleted,spiked cell samples were also stained with fluorescent host isotypecontrols (mouse IgG1:PE and mouse IgG2b:APC) to determine backgroundlevels of cellular staining, known to be high in most lymphocytes.Undepleted, unstained spiked cells were also prepared as an additionalbackground control.

Step 7) Flow cytometric analysis. Samples were analyzed using an LSRIIcytometer equipped with appropriate lasers and fluorescent emissiondetectors. 10,000 events were recorded per sample. Raw data files (.fcs)were exported to FlowJo software (TreeStar, Ashland, Oreg.). 2D ‘dotplots’ were visualized as PE signal vs. APC signal. Quadrants were gatedbased on isotype-control background levels (data not shown). Her2+ cellswere defined as those having relative fluorescence intensity (RFI)>8000PE units. EpCAM+ cells were defined as those having RFI>1000 APC units.Her2+/EpCAM+ cells were defined as “tumor cells” meeting bothfluorescence intensity criteria. Her2−/EpCAM− cells were defined as“leukocytes” meeting neither positive criteria. Experimental results areshown in FIG. 81 and Table 19.

TABLE 19 Her2+ Her2− leukocytes: depletion Panel depletion conditionsEpCAM+ % D EpCAM− % D tumor cells ratio A undepleted 9.95 n/a 68.5 n/a6.9 n/a B −HyLk1′ 12.6 26.6 71.3 4.1 5.7 0.8 C −Herceptin −HyLk1′ 12.121.6 74.9 9.3 6.2 0.9 D conventional method 4.4 −56.0 80.2 17.1 18.3 2.7E modified conventional 6.8 −32.2 82.8 20.9 12.3 1.8 F HybriLink method2.2 −77.9 85.9 25.4 39.0 5.7

In the following, further embodiments are explained with the help ofsubsequent examples starting at Example 20A.

Example 20A

A method for detecting one or more biological targets of a sample in adetection assay, comprising: i) providing a molecular probe, comprisinga binding moiety and an oligonucleotide sequence, to a sample comprisingone or more biological targets; ii) binding the one or more biologicaltargets with the binding moiety; iii) providing a detectable componentto the sample, wherein the detectable component comprises a signalgenerating moiety conjugated to an oligonucleotide sequencecomplementary to the oligonucleotide sequence of the molecular probe; v)hydridizing the oligonucleotide sequence of the target-bound molecularprobe to the detectable component; and v) detecting a signal generatedfrom the hydridized detectable component; wherein: a) the conjugationbetween the oligonucleotide sequence and the binding moiety andconjugation between the complementary oligonucleotide sequence and thesignal generating moiety, comprise a covalent bond linkage, comprising ahydrazone, oxime, triazine, or other bond, wherein the formation of theconjugates are at least 90% efficient; b) the binding moiety comprises abinding affinity of less than 10⁻⁴ M for the one or more biologicaltargets.

Example 21A

The method of Example 20A, wherein sample is a complex sample.

Example 22A

The method of one or more of Examples 20A-21A, wherein the hydridizationefficiency of the oligonucleotide sequence to the complementaryoligonucleotide sequence is at least 50%.

Example 23A

The method of one or more of Examples 20A-22A, wherein the molecularprobe has substantially the same solubility as the binding moiety.

Example 24A

The method of any one of Examples 20A-23A, wherein the molecular probecomprises a molecular weight of between about 15,000 Daltons to about450,000 Daltons.

Example 25A

The method of one or more of Examples 20A-24A, wherein the method ofdetection generates less false positives than secondary antibodydetection methods.

Example 26A

The method of one or more of Examples 20A-25A, wherein the methodfurther comprises: i) preparing: a) a molecular probe, comprising abinding moiety conjugated to an oligonucleotide sequence; and b) adetectable component, comprising a signal generating moiety conjugatedto an oligonucleotide sequence complementary to the oligonucleotidesequence of the molecular probe; wherein the prepared molecular probeand prepared detectable component have at least 90% purity.

Example 27A

The method of one or more of Examples 20A-26A, wherein the solubility ofthe molecular probe minimizes non-specific binding to the one or morebiological targets.

Example 28A

The method of one or more of Examples 20A-27A, wherein the sample ishomogeneous or heterogenous mixture;

Example 29A

The method of one or more of Examples 20A-28A, wherein the samplecomprises biological fluids or fluidized biological tissue.

Example 30A

The method of one or more of Examples 20A-29A, wherein the samplecomprises cells, membranes, biological molecules, metabolites, ordisease biomarkers.

Example 31A

The method of one or more of Examples 20A-30A, wherein the samplecomprises a range of analytes having a wide range of bindingspecificities.

Example 32A

The method of one or more of Examples 20A-31A, wherein the time ofconducting the method, from the start of preparation to the end ofdetection is between about 2-3 hours.

Example 33A

The method of one or more of Examples 20A-32A, wherein the molecularprobe is neutral in charge.

Example 34A

The method of one or more of Examples 20A-33A, wherein the methodfurther comprises preparing and isolating a molecular probe, comprisinga binding moiety conjugated to an oligonucleotide sequence, comprising:i) introducing a modified binding moiety into a buffered solution;

ii) conjugating the modified binding moieties with at least one modifiedoligonucleotide at greater than 90% efficiency to form bindingmoiety-oligonucleotide conjugates; and iii) isolating the bindingmoiety-oligonucleotide conjugates from the conjugation solution bybinding the conjugates to an immobilized binder, removing theunconjugated oligonucleotide in a wash step followed by release of thebound conjugate from the solid support.

Example 35A

The method of one or more of Examples 20A-34A, wherein the methodfurther comprises preparing and isolating a detectable component,comprising a signal generating moiety conjugated to an oligonucleotidesequence complementary to the oligonucleotide sequence of the molecularprobe, comprising: i) introducing a modified signal generating moietyinto a buffered solution; ii) conjugating the modified signal generatingmoieties with at least one modified complementary oligonucleotide atgreater than 90% efficiency to form signal generatingmoiety-complementary oligonucleotide conjugates; and iii) isolating thesignal generating moiety-complementary oligonucleotide conjugates fromthe conjugation solution by binding the conjugates to an immobilizedbinder, removing the unconjugated oligonucleotide in a wash stepfollowed by release of the bound conjugate from the solid support.

Example 36A

The method of one or more of Examples 20A-35A, wherein the methodfurther comprises preparing and isolating a detectable component,comprising a scaffold, comprising one or more signal generatingmoieties, conjugated to an oligonucleotide sequence complementary to theoligonucleotide sequence of the molecular probe, comprising: i)introducing a modified scaffold, comprising the one or more signalgenerating moieties, into a buffered solution; ii) conjugating themodified scaffold with at least one modified complementaryoligonucleotide at greater than 90% efficiency to formscaffold-complementary oligonucleotide conjugates; and iii) isolatingthe scaffold-complementary oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support.

Example 37A

The method of one or more of Examples 20A-36A, wherein the scaffoldcomprises a dendrimer, polysaccharide, or a dextran.

Example 38A

The method of one or more of Examples 20A-37A, wherein the one or moremolecular probes comprise a range of specificities for the one or morebiological targets.

Example 39A

The method of one or more of Examples 20A-38A, wherein the detectionassay comprises a singleplex or multiplex detection assay, comprisingimmunodetection, flow cytometry, immunohistochemistry, microspcopy,imaging, high content screening (HCS), ELISA, ELISpot, arrays, beadarrays, or combinations or derivatives thereof.

Example 40A

The method of one or more of Examples 20A-39A, wherein the bindingmoiety comprises an antibody, a protein, a peptide, a carbohydrate, anuclear receptor, a small molecule, or combinations or derivativesthereof.

Example 41A

The method of one or more of Examples 20A-40A, wherein the one or morebiological target comprises an antigen, a pathogen, a protein, apeptide, an epitope, a carbohydrate-containing molecule, a smallmolecule, or combinations or derivatives thereof.

Example 42A

The method of one or more of Examples 20A-41A, wherein the one or moresignal generating moieties, comprise one or more fluorophors,biofluorescent proteins, quantum dots, Raman particles, or combinationsor derivatives thereof.

Example 43A

The method of one or more of Examples 20A-42A, wherein the one or moresignal generating moieties provides an enhanced signal that minimizesdetection errors from background noise.

Example 44A

The method of one or more of Examples 20A-43A, wherein the one or moremolecular probes and/or the one or more detectable components furthercomprise a spacer group, comprising a polymerized ethylene oxide, a PEG,or a PEO.

Example 45A

The method of one or more of Examples 20A-44A, wherein the modifiedbinding moiety, the modified scaffold, the oligonucleotide sequence,and/or the complementary oligonucleotide sequence comprise HyNic or4-FB.

Example 46A

The method of one or more of Examples 20A-45A, wherein the one or moremolecular probes comprise unique, distinguishable, and/or specificallydesigned oligonucleotide sequences.

Example 47A

The method of one or more of Examples 20A-46A, wherein the one or moredetectable components comprise unique, distinguishable, and/orspecifically designed complementary oligonucleotide sequences.

Example 48A

The method of one or more of Examples 20A-47A, wherein theoligonucleotide sequences of the one or more molecular probes areuniquely and specifically designed to hybridize to the complementaryoligonucleotide sequence of the one or more detectable components.

Example 49A

The method of one or more of Examples 20A-48A, wherein theoligonucleotide sequences and/or complementary oligonucleotidesequences, comprise 3′-oligonucleotides, 5′-oligonucleotides, LNAs,PNAs, or combinations or derivatives thereof.

Example 50A

The method of one or more of Examples 20A-49A, wherein the methodfurther comprises: i) provides a universal adapter to the complexsample, wherein the universal adapter comprised an oligonucleotidesequence having a first sequence segment complementary to theoligonucleotide sequence of the molecular probe and a second sequencesegment complementary to the oligonucleotide sequence of the detectablecomponent; ii) hydridizing the oligonucleotide sequences of the one ormore target-bound molecular probes to the first oligonucleotide sequencesegment of the universal adapter; iii) providing the one or moredetectable components to the complex sample; iv) hydridizing theoligonucleotide sequences of the one or more detectable components tothe second oligonucleotide sequence segment of the universal adapter;and v) detecting one or more signals generated from the hydridized oneor more detectable components.

Example 51A

The method of one or more of Examples 20A-50A, wherein the methodfurther comprises: i) providing at least a first molecular probe and asecond molecular probe, comprising a first binding moiety conjugated toa first oligonucleotide sequence and a second binding moiety conjugatedto a second oligonucleotide sequence, respectively, to the complexsample comprising the one or more biological targets; ii) specificallybinding the one or more biological targets with the binding moiety ofthe first molecular probe and the binding moiety of the second molecularprobe; iii) providing the one or more detectable components to thecomplex sample; iv) hydridizing the first oligonucleotide sequence ofthe first target-bound molecular probe to a complementaryoligonucleotide sequence conjugated to a bead; v) hydridizing the secondoligonucleotide sequence of the second target-bound molecular probe tothe complementary oligonucleotide sequences of the one or moredetectable components; and vi) detecting one or more signals generatedfrom the one or more hydridized detectable components.

Example 52A

The method of one or more of Examples 20A-51A, wherein the methodfurther comprises i) providing at least a first molecular probe and asecond molecular probe, comprising a first binding moiety conjugated toa first oligonucleotide sequence and a second binding moiety conjugatedto a second oligonucleotide sequence, respectively, to the complexsample comprising the one or more biological targets; ii) specificallybinding the one or more biological targets with the binding moiety ofthe first molecular probe and the binding moiety of the second molecularprobe; iii) providing a universal adapter to the complex sample, whereinthe universal adapter comprised an oligonucleotide sequence having afirst sequence segment complementary to the oligonucleotide sequence ofthe molecular probe and a second sequence segment complementary to theoligonucleotide sequence of the detectable component; iv) hydridizingthe first oligonucleotide sequence of the first target-bound molecularprobe to a first portion of the first oligonucleotide sequence segmentof the universal adapter; v) hydridizing the second oligonucleotidesequence of the second target-bound molecular probe to a second portionof the first oligonucleotide sequence segment of the universal adapter;vi) providing the one or more detectable components to the complexsample; vii) hydridizing the oligonucleotide sequences of the one ormore detectable components to the first and second portions of thesecond oligonucleotide sequence segment of the universal adapter; andviii) detecting one or more signals generated from the hydridized one ormore detectable components.

Example 53A

A method for detecting one or more biological targets in a detectionassay, comprising: i) providing at least a first and a second molecularprobe, each comprising a binding moiety conjugated to an oligonucleotidesequence, to a sample comprising the one or more biological targets; ii)binding the one or more biological targets with the binding moiety ofthe first molecular probe and the binding moiety of the second molecularprobe; iii) optionally hydridizing the oligonucleotide sequence of thefirst target-bound molecular probe to a complementary oligonucleotidesequence conjugated to a bead; iv) hydridizing the oligonucleotidesequence of the second target-bound molecular probe to a complementaryoligonucleotide sequence conjugated to a detectable component; and v)detecting a signal generated from the hydridized detectable component;wherein: a) each binding moiety has a binding affinity for the one ormore biological targets; and b) the conjugation between the respectiveoligonucleotide or complementary oligonucleotide sequences and thebinding moiety of the first molecular probe, the binding moiety of thesecond molecular probe, the bead, or the detectable component, comprisesa covalent bond linkage, comprising a hydrazone, oxime, triazine, orother bond, wherein the formation of the conjugate is at least 90%efficient.

Example 54A

A method for detecting one or more biological targets in a detectionassay, comprising: i) providing a first molecular probe, comprising afirst binding moiety conjugated to a first oligonucleotide sequence, toa sample comprising the one or more biological targets; ii) binding theone or more biological targets via the first binding moiety of the firstmolecular probe; iii) providing a second molecular probe, comprising asecond binding moiety conjugated to a second oligonucleotide sequence,to the sample comprising the one or more biological targets; iv) bindingthe one or more biological targets via the second binding moiety of thesecond molecular probe; v) optionally hydridizing the firstoligonucleotide sequence of the first target-bound molecular probe to acomplementary oligonucleotide sequence conjugated to a bead; vi)hydridizing the second oligonucleotide sequence of the secondtarget-bound molecular probe to a complementary oligonucleotide sequenceconjugated to a detectable component; and vii) detecting a signalgenerated from the hydridized detectable component; wherein: a) eachbinding moiety has a binding affinity for the one or more biologicaltargets; b) the conjugation between the respective oligonucleotide orcomplementary oligonucleotide sequences and the first binding moiety,the second binding moiety, the bead, or the detectable component,comprises a covalent bond linkage, comprising a hydrazone, oxime,triazine, or other bond, wherein the formation of the conjugate is atleast 90% efficient.

Example 55A

A method for detecting one or more biological targets of a sample in adetection assay, comprising: i) providing a molecular probe, comprisinga binding moiety and an oligonucleotide sequence, to a sample comprisingone or more biological targets; ii) binding the one or more biologicaltargets with the binding moiety; iii) providing a detectable componentto the sample, wherein the detectable component comprises a signalgenerating moiety conjugated to an oligonucleotide sequencecomplementary to the oligonucleotide sequence of the molecular probe;iv) hydridizing the oligonucleotide sequence of the target-boundmolecular probe to the detectable component; and v) detecting a signalgenerated from the hydridized detectable component; wherein: a) theconjugation between the oligonucleotide sequence and the binding moietyand conjugation between the complementary oligonucleotide sequence andthe signal generating moiety, comprise a covalent bond linkage,comprising a hydrazone, oxime, triazine, or other bond, wherein theformation of the conjugates are at least 90% efficient; b) the bindingmoiety comprises a binding affinity of less than 10⁻⁴ M for the one ormore biological targets.

Example 56A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) providing a molecular probe,comprising a binding moiety conjugated to an oligonucleotide sequence,to the complex sample comprising the one or more biological targets; ii)specifically binding the one or more biological targets with the bindingmoiety; iii) providing a detectable component to the complex sample,wherein the detectable component comprising a signal generating moietyconjugated to an oligonucleotide sequence complementary to theoligonucleotide sequence of the molecular probe; iv) hydridizing theoligonucleotide sequence of the target-bound molecular probe to thecomplementary oligonucleotide sequence of the detectable component; andv) detecting a signal generated from the hydridized detectablecomponent; wherein: a) the conjugation between the oligonucleotidesequence and the binding moiety and conjugation between thecomplementary oligonucleotide sequence and the signal generating moiety,comprises a covalent bond linkage, comprising a hydrazone, oxime,triazine, or other bond, wherein the formation of the conjugates are atleast 90% efficient; b) the binding moiety comprises a binding affinityof less than 10⁻⁴ M for the one or more biological targets; and c) thehydridization efficiency of the oligonucleotide sequence to thecomplementary oligonucleotide sequence is at least 50%.

Example 57A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) providing a molecular probe,comprising a binding moiety conjugated to an oligonucleotide sequence,to the complex sample comprising the one or more biological targets; ii)specifically binding the one or more biological targets with the bindingmoiety; iii) providing a detectable component to the complex sample,wherein the detectable component comprising a signal generating moietyconjugated to an oligonucleotide sequence complementary to theoligonucleotide sequence of the molecular probe; iv) hydridizing theoligonucleotide sequence of the target-bound molecular probe to thecomplementary oligonucleotide sequence of the detectable component; andv) detecting a signal generated from the hydridized detectablecomponent; wherein: a) the conjugation between the oligonucleotidesequence and the binding moiety and conjugation between thecomplementary oligonucleotide sequence and the signal generating moiety,comprises a covalent bond linkage, comprising a hydrazone, oxime,triazine, or other bond, wherein the formation of the conjugates are atleast 90% efficient; b) the binding moiety comprises a binding affinityof less than 10⁻⁴ M for the one or more biological targets; c) thehydridization efficiency of the oligonucleotide sequence to thecomplementary oligonucleotide sequence is at least 50%; and d) themolecular probe has substantially the same solubility as the bindingmoiety.

Example 58A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) providing a molecular probe,comprising a binding moiety conjugated to an oligonucleotide sequence,to the complex sample comprising the one or more biological targets; ii)specifically binding the one or more biological targets with the bindingmoiety; iii) providing a detectable component to the complex sample,wherein the detectable component comprising a signal generating moietyconjugated to an oligonucleotide sequence complementary to theoligonucleotide sequence of the molecular probe; iv) hydridizing theoligonucleotide sequence of the target-bound molecular probe to thecomplementary oligonucleotide sequence of the detectable component; andv) detecting a signal generated from the hydridized detectablecomponent; wherein: a) the conjugation between the oligonucleotidesequence and the binding moiety and conjugation between thecomplementary oligonucleotide sequence and the signal generating moiety,comprises a covalent bond linkage, comprising a hydrazone, oxime,triazine, or other bond, wherein the formation of the conjugates are atleast 90% efficient; b) the binding moiety comprises a binding affinityof less than 10⁻⁴ M for the one or more biological targets; c) thehydridization efficiency of the oligonucleotide sequence to thecomplementary oligonucleotide sequence is at least 50%; d) the molecularprobe has substantially the same solubility as the binding moiety; ande) the molecular probe comprises a molecular weight of between about15,000 Daltons to about 450,000 Daltons.

Example 59A

In certain embodiments, the method for detecting one or more biologicaltargets of a complex sample in a detection assay, comprising: i)providing a molecular probe, comprising a binding moiety conjugated toan oligonucleotide sequence, to the complex sample comprising the one ormore biological targets; ii) specifically binding the one or morebiological targets with the binding moiety; iii) providing a detectablecomponent to the complex sample, wherein the detectable componentcomprising a signal generating moiety conjugated to an oligonucleotidesequence complementary to the oligonucleotide sequence of the molecularprobe; iv) hydridizing the oligonucleotide sequence of the target-boundmolecular probe to the complementary oligonucleotide sequence of thedetectable component; and v) detecting a signal generated from thehydridized detectable component; wherein: a) the conjugation between theoligonucleotide sequence and the binding moiety and conjugation betweenthe complementary oligonucleotide sequence and the signal generatingmoiety, comprises a covalent bond linkage, comprising a hydrazone,oxime, triazine, or other bond, wherein the formation of the conjugatesare at least 90% efficient; b) the binding moiety comprises a bindingaffinity of less than 10⁻⁴ M for the one or more biological targets; c)the hydridization efficiency of the oligonucleotide sequence to thecomplementary oligonucleotide sequence is at least 50%; d) the molecularprobe has substantially the same solubility as the binding moiety; e)the molecular probe comprises a molecular weight of between about 15,000Daltons to about 450,000 Daltons; and f) the method of detectiongenerates less false positives than secondary antibody detectionmethods.

Example 60A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) preparing: a) a molecularprobe, comprising a binding moiety conjugated to an oligonucleotidesequence; and b) a detectable component, comprising a signal generatingmoiety conjugated to an oligonucleotide sequence complementary to theoligonucleotide sequence of the molecular probe; ii) providing themolecular probe to the complex sample comprising the one or morebiological targets; iii) specifically binding the one or more biologicaltargets with the binding moiety; iv) providing the detectable componentto the complex sample; v) hydridizing the oligonucleotide sequence ofthe target-bound molecular probe to the complementary oligonucleotidesequence of the detectable component; and vi) detecting a signalgenerated from the hydridized detectable component; wherein: a) theconjugation between the oligonucleotide sequence and the binding moietyand conjugation between the complementary oligonucleotide sequence andthe signal generating moiety, comprises a covalent bond linkage,comprising a hydrazone, oxime, triazine, or other bond, wherein theformation of the conjugates are at least 90% efficient; b) the bindingmoiety comprises a binding affinity of less than 10⁻⁴ M for the one ormore biological targets; c) the hydridization efficiency of theoligonucleotide sequence to the complementary oligonucleotide sequenceis at least 50%; d) the molecular probe has substantially the samesolubility as the binding moiety; e) the molecular probe comprises amolecular weight of between about 15,000 Daltons to about 450,000Daltons; f) the method of detection generates less false positives thansecondary antibody detection methods; and g) the prepared molecularprobe and prepared detectable component have at least 90% purity.

Example 61A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) preparing: a) a molecularprobe, comprising a binding moiety conjugated to an oligonucleotidesequence; and b) a detectable component, comprising a signal generatingmoiety conjugated to an oligonucleotide sequence complementary to theoligonucleotide sequence of the molecular probe; ii) providing themolecular probe to the complex sample comprising the one or morebiological targets; iii) specifically binding the one or more biologicaltargets with the binding moiety; iv) providing the detectable componentto the complex sample; v) hydridizing the oligonucleotide sequence ofthe target-bound molecular probe to the complementary oligonucleotidesequence of the detectable component; and vi) detecting a signalgenerated from the hydridized detectable component; wherein: a) theconjugation between the oligonucleotide sequence and the binding moietyand conjugation between the complementary oligonucleotide sequence andthe signal generating moiety, comprises a covalent bond linkage,comprising a hydrazone, oxime, triazine, or other bond, wherein theformation of the conjugates are at least 90% efficient; b) the bindingmoiety comprises a binding affinity of less than 10⁻⁴ M for the one ormore biological targets; c) the hydridization efficiency of theoligonucleotide sequence to the complementary oligonucleotide sequenceis at least 50%; d) the molecular probe has substantially the samesolubility as the binding moiety; e) the molecular probe comprises amolecular weight of between about 15,000 Daltons to about 450,000Daltons; f) the method of detection generates less false positives thansecondary antibody detection methods; g) the prepared molecular probeand prepared detectable component have at least 90% purity; h) thesolubility of the molecular probe minimizes non-specific binding to theone or more biological targets.

Example 62A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) preparing: a) a molecularprobe, comprising a binding moiety conjugated to an oligonucleotidesequence; and b) a detectable component, comprising a signal generatingmoiety conjugated to an oligonucleotide sequence complementary to theoligonucleotide sequence of the molecular probe; ii) providing themolecular probe to the complex sample comprising the one or morebiological targets; iii) specifically binding the one or more biologicaltargets with the binding moiety; iv) providing the detectable componentto the complex sample; v) hydridizing the oligonucleotide sequence ofthe target-bound molecular probe to the complementary oligonucleotidesequence of the detectable component; and vi) detecting a signalgenerated from the hydridized detectable component; wherein: a) theconjugation between the oligonucleotide sequence and the binding moietyand conjugation between the complementary oligonucleotide sequence andthe signal generating moiety, comprises a covalent bond linkage,comprising a hydrazone, oxime, triazine, or other bond, wherein theformation of the conjugates are at least 90% efficient; b) the bindingmoiety comprises a binding affinity of less than 10⁻⁴ M for the one ormore biological targets; c) the hydridization efficiency of theoligonucleotide sequence to the complementary oligonucleotide sequenceis at least 50%; d) the molecular probe has substantially the samesolubility as the binding moiety; e) the molecular probe comprises amolecular weight of between about 15,000 Daltons to about 450,000Daltons; f) the method of detection generates less false positives thansecondary antibody detection methods; g) the prepared molecular probeand prepared detectable component have at least 90% purity; h) thesolubility of the molecular probe minimizes non-specific binding to theone or more biological targets; and i) the sample is homogeneous orheterogenous mixture.

Example 63A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) preparing: a) a molecularprobe, comprising a binding moiety conjugated to an oligonucleotidesequence; and b) a detectable component, comprising a signal generatingmoiety conjugated to an oligonucleotide sequence complementary to theoligonucleotide sequence of the molecular probe; ii) providing themolecular probe to the complex sample comprising the one or morebiological targets; iii) specifically binding the one or more biologicaltargets with the binding moiety; iv) providing the detectable componentto the complex sample; v) hydridizing the oligonucleotide sequence ofthe target-bound molecular probe to the complementary oligonucleotidesequence of the detectable component; and vi) detecting a signalgenerated from the hydridized detectable component; wherein: a) theconjugation between the oligonucleotide sequence and the binding moietyand conjugation between the complementary oligonucleotide sequence andthe signal generating moiety, comprises a covalent bond linkage,comprising a hydrazone, oxime, triazine, or other bond, wherein theformation of the conjugates are at least 90% efficient; b) the bindingmoiety comprises a binding affinity of less than 10⁻⁴ M for the one ormore biological targets; c) the hydridization efficiency of theoligonucleotide sequence to the complementary oligonucleotide sequenceis at least 50%; d) the molecular probe has substantially the samesolubility as the binding moiety; e) the molecular probe comprises amolecular weight of between about 15,000 Daltons to about 450,000Daltons; f) the method of detection generates less false positives thansecondary antibody detection methods; g) the prepared molecular probeand prepared detectable component have at least 90% purity; h) thesolubility of the molecular probe minimizes non-specific binding to theone or more biological targets; i) the sample is homogeneous orheterogenous mixture; and j) the sample comprises biological fluids orfluidized biological tissue.

Example 64A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) preparing: a) a molecularprobe, comprising a binding moiety conjugated to an oligonucleotidesequence; and b) a detectable component, comprising a signal generatingmoiety conjugated to an oligonucleotide sequence complementary to theoligonucleotide sequence of the molecular probe; ii) providing themolecular probe to the complex sample comprising the one or morebiological targets; iii) specifically binding the one or more biologicaltargets with the binding moiety; iv) providing the detectable componentto the complex sample; v) hydridizing the oligonucleotide sequence ofthe target-bound molecular probe to the complementary oligonucleotidesequence of the detectable component; and vi) detecting a signalgenerated from the hydridized detectable component; wherein: a) theconjugation between the oligonucleotide sequence and the binding moietyand conjugation between the complementary oligonucleotide sequence andthe signal generating moiety, comprises a covalent bond linkage,comprising a hydrazone, oxime, triazine, or other bond, wherein theformation of the conjugates are at least 90% efficient; b) the bindingmoiety comprises a binding affinity of less than 10⁻⁴ M for the one ormore biological targets; c) the hydridization efficiency of theoligonucleotide sequence to the complementary oligonucleotide sequenceis at least 50%; d) the molecular probe has substantially the samesolubility as the binding moiety; e) the molecular probe comprises amolecular weight of between about 15,000 Daltons to about 450,000Daltons; f) the method of detection generates less false positives thansecondary antibody detection methods; g) the prepared molecular probeand prepared detectable component have at least 90% purity; h) thesolubility of the molecular probe minimizes non-specific binding to theone or more biological targets; i) the sample is homogeneous orheterogenous mixture; j) the sample comprises biological fluids orfluidized biological tissue; and k) the sample comprises cells,membranes, biological molecules, metabolites, or disease biomarkers.

Example 65A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) preparing: a) a molecularprobe, comprising a binding moiety conjugated to an oligonucleotidesequence; and b) a detectable component, comprising a signal generatingmoiety conjugated to an oligonucleotide sequence complementary to theoligonucleotide sequence of the molecular probe; ii) providing themolecular probe to the complex sample comprising the one or morebiological targets; iii) specifically binding the one or more biologicaltargets with the binding moiety; iv) providing the detectable componentto the complex sample; v) hydridizing the oligonucleotide sequence ofthe target-bound molecular probe to the complementary oligonucleotidesequence of the detectable component; and vi) detecting a signalgenerated from the hydridized detectable component; wherein: a) theconjugation between the oligonucleotide sequence and the binding moietyand conjugation between the complementary oligonucleotide sequence andthe signal generating moiety, comprises a covalent bond linkage,comprising a hydrazone, oxime, triazine, or other bond, wherein theformation of the conjugates are at least 90% efficient; b) the bindingmoiety comprises a binding affinity of less than 10⁻⁴ M for the one ormore biological targets; c) the hydridization efficiency of theoligonucleotide sequence to the complementary oligonucleotide sequenceis at least 50%; d) the molecular probe has substantially the samesolubility as the binding moiety; e) the molecular probe comprises amolecular weight of between about 15,000 Daltons to about 450,000Daltons; 1) the method of detection generates less false positives thansecondary antibody detection methods; g) the prepared molecular probeand prepared detectable component have at least 90% purity; h) thesolubility of the molecular probe minimizes non-specific binding to theone or more biological targets; i) the sample is homogeneous orheterogenous mixture; j) the sample comprises biological fluids orfluidized biological tissue; k) the sample comprises cells, membranes,biological molecules, metabolites, or disease biomarkers; and l) thesample comprises a range of analytes having a wide range of bindingspecificities.

Example 66A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) preparing: a) a molecularprobe, comprising a binding moiety conjugated to an oligonucleotidesequence; and b) a detectable component, comprising a signal generatingmoiety conjugated to an oligonucleotide sequence complementary to theoligonucleotide sequence of the molecular probe; ii) providing themolecular probe to the complex sample comprising the one or morebiological targets; iii) specifically binding the one or more biologicaltargets with the binding moiety; iv) providing the detectable componentto the complex sample; v) hydridizing the oligonucleotide sequence ofthe target-bound molecular probe to the complementary oligonucleotidesequence of the detectable component; and vi) detecting a signalgenerated from the hydridized detectable component; wherein: a) theconjugation between the oligonucleotide sequence and the binding moietyand conjugation between the complementary oligonucleotide sequence andthe signal generating moiety, comprises a covalent bond linkage,comprising a hydrazone, oxime, triazine, or other bond, wherein theformation of the conjugates are at least 90% efficient; b) the bindingmoiety comprises a binding affinity of less than 10⁻⁴ M for the one ormore biological targets; c) the hydridization efficiency of theoligonucleotide sequence to the complementary oligonucleotide sequenceis at least 50%; d) the molecular probe has substantially the samesolubility as the binding moiety; e) the molecular probe comprises amolecular weight of between about 15,000 Daltons to about 450,000Daltons; f) the method of detection generates less false positives thansecondary antibody detection methods; g) the prepared molecular probeand prepared detectable component have at least 90% purity; h) thesolubility of the molecular probe minimizes non-specific binding to theone or more biological targets; i) the sample is homogeneous orheterogenous mixture; j) the sample comprises biological fluids orfluidized biological tissue; k) the sample comprises cells, membranes,biological molecules, metabolites, or disease biomarkers; l) the samplecomprises a range of analytes having a wide range of bindingspecificities; and m) the time of conducting the method, from the startof preparation to the end of detection is between about 2-3 hours.

Example 67A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) preparing: a) a molecularprobe, comprising a binding moiety conjugated to an oligonucleotidesequence; and b) a detectable component, comprising a signal generatingmoiety conjugated to an oligonucleotide sequence complementary to theoligonucleotide sequence of the molecular probe; ii) providing themolecular probe to the complex sample comprising the one or morebiological targets; iii) specifically binding the one or more biologicaltargets with the binding moiety; iv) providing the detectable componentto the complex sample; v) hydridizing the oligonucleotide sequence ofthe target-bound molecular probe to the complementary oligonucleotidesequence of the detectable component; and vi) detecting a signalgenerated from the hydridized detectable component; wherein: a) theconjugation between the oligonucleotide sequence and the binding moietyand conjugation between the complementary oligonucleotide sequence andthe signal generating moiety, comprises a covalent bond linkage,comprising a hydrazone, oxime, triazine, or other bond, wherein theformation of the conjugates are at least 90% efficient; b) the bindingmoiety comprises a binding affinity of less than 10⁻⁴ M for the one ormore biological targets; c) the hydridization efficiency of theoligonucleotide sequence to the complementary oligonucleotide sequenceis at least 50%; d) the molecular probe has substantially the samesolubility as the binding moiety; e) the molecular probe comprises amolecular weight of between about 15,000 Daltons to about 450,000Daltons; f) the method of detection generates less false positives thansecondary antibody detection methods; g) the prepared molecular probeand prepared detectable component have at least 90% purity; h) thesolubility of the molecular probe minimizes non-specific binding to theone or more biological targets; i) the sample is homogeneous orheterogenous mixture; j) the sample comprises biological fluids orfluidized biological tissue; k) the sample comprises cells, membranes,biological molecules, metabolites, or disease biomarkers; l) the samplecomprises a range of analytes having a wide range of bindingspecificities; m) the time of conducting the method, from the start ofpreparation to the end of detection is between about 2-3 hours; and n)the molecular probe is neutral in charge.

Example 68A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) preparing and isolating amolecular probe, comprising a binding moiety conjugated to anoligonucleotide sequence, comprising: a) introducing a modified bindingmoiety into a buffered solution; b) conjugating the modified bindingmoieties with at least one modified oligonucleotide at greater than 90%efficiency to form binding moiety-oligonucleotide conjugates; and c)isolating the binding moiety-oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and ii) preparingand isolating a detectable component, comprising a signal generatingmoiety conjugated to an oligonucleotide sequence complementary to theoligonucleotide sequence of the molecular probe, comprising: a)introducing a modified signal generating moiety into a bufferedsolution; b) conjugating the modified signal generating moieties with atleast one modified complementary oligonucleotide at greater than 90%efficiency to form signal generating moiety-complementaryoligonucleotide conjugates; and c) isolating the signal generatingmoiety-complementary oligonucleotide conjugates from the conjugationsolution by binding the conjugates to an immobilized binder, removingthe unconjugated oligonucleotide in a wash step followed by release ofthe bound conjugate from the solid support; and iii) providing themolecular probe to the complex sample comprising the one or morebiological targets; iv) specifically binding the one or more biologicaltargets with the binding moiety; v) providing the detectable componentto the complex sample; vi) hydridizing the oligonucleotide sequence ofthe target-bound molecular probe to the complementary oligonucleotidesequence of the detectable component; and vii) detecting a signalgenerated from the hydridized detectable component; wherein: a) theconjugation between the oligonucleotide sequence and the binding moietyand conjugation between the complementary oligonucleotide sequence andthe signal generating moiety, comprises a covalent bond linkage,comprising a hydrazone, oxime, triazine, or other bond, wherein theformation of the conjugates are at least 90% efficient; b) the bindingmoiety comprises a binding affinity of less than 10⁻⁴ M for the one ormore biological targets; c) the hydridization efficiency of theoligonucleotide sequence to the complementary oligonucleotide sequenceis at least 50%; d) the molecular probe has substantially the samesolubility as the binding moiety; e) the molecular probe comprises amolecular weight of between about 15,000 Daltons to about 450,000Daltons; f) the method of detection generates less false positives thansecondary antibody detection methods; g) the prepared and isolatedmolecular probe and prepared and isolated detectable component have atleast 90% purity; h) the solubility of the molecular probe minimizesnon-specific binding to the one or more biological targets; i) thesample is homogeneous or heterogenous mixture; j) the sample comprisesbiological fluids or fluidized biological tissue; k) the samplecomprises cells, membranes, biological molecules, metabolites, ordisease biomarkers; l) the sample comprises a range of analytes having awide range of binding specificities; m) the time of conducting themethod, from the start of preparation to the end of detection is betweenabout 2-3 hours; and n) the molecular probe is neutral in charge.

Example 69A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) preparing and isolating amolecular probe, comprising a binding moiety conjugated to anoligonucleotide sequence, comprising: a) introducing a modified bindingmoiety into a buffered solution; b) conjugating the modified bindingmoieties with at least one modified oligonucleotide at greater than 90%efficiency to form binding moiety-oligonucleotide conjugates; and c)isolating the binding moiety-oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and ii) preparingand isolating a detectable component, comprising a scaffold, comprisingone or more signal generating moieties, conjugated to an oligonucleotidesequence complementary to the oligonucleotide sequence of the molecularprobe, comprising: a) introducing a modified scaffold, comprising theone or more signal generating moieties, into a buffered solution; b)conjugating the modified scaffold with at least one modifiedcomplementary oligonucleotide at greater than 90% efficiency to formscaffold-complementary oligonucleotide conjugates; and c) isolating thescaffold-complementary oligonucleotide conjugates from the conjugationsolution by binding the conjugates to an immobilized binder, removingthe unconjugated oligonucleotide in a wash step followed by release ofthe bound conjugate from the solid support; and iii) providing themolecular probe to the complex sample comprising the one or morebiological targets; iv) specifically binding the one or more biologicaltargets with the binding moiety; v) providing the detectable componentto the complex sample; vi) hydridizing the oligonucleotide sequence ofthe target-bound molecular probe to the complementary oligonucleotidesequence of the detectable component; and vii) detecting a signalgenerated from the hydridized detectable component; wherein: a) theconjugation between the oligonucleotide sequence and the binding moietyand conjugation between the complementary oligonucleotide sequence andthe scaffold, comprises a covalent bond linkage, comprising a hydrazone,oxime, triazine, or other bond, wherein the formation of the conjugatesare at least 90% efficient; b) the binding moiety comprises a bindingaffinity of less than 10⁻⁴ M for the one or more biological targets; c)the hydridization efficiency of the oligonucleotide sequence to thecomplementary oligonucleotide sequence is at least 50%; d) the molecularprobe has substantially the same solubility as the binding moiety; e)the molecular probe comprises a molecular weight of between about 15,000Daltons to about 450,000 Daltons; f) the method of detection generatesless false positives than secondary antibody detection methods; g) theprepared and isolated molecular probe and prepared and isolateddetectable component have at least 90% purity; h) the solubility of themolecular probe minimizes non-specific binding to the one or morebiological targets; i) the sample is homogeneous or heterogenousmixture; j) the sample comprises biological fluids or fluidizedbiological tissue; k) the sample comprises cells, membranes, biologicalmolecules, metabolites, or disease biomarkers; l) the sample comprises arange of analytes having a wide range of binding specificities; m) thetime of conducting the method, from the start of preparation to the endof detection is between about 2-3 hours; n) the molecular probe isneutral in charge; and o) the scaffold comprises a dendrimer,polysaccharide, or a dextran.

Example 70A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) preparing and isolating oneor more molecular probes, comprising a binding moiety conjugated to anoligonucleotide sequence, comprising: a) introducing a modified bindingmoiety into a buffered solution; b) conjugating the modified bindingmoieties with at least one modified oligonucleotide at greater than 90%efficiency to form binding moiety-oligonucleotide conjugates; and c)isolating the binding moiety-oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and ii) preparingand isolating one or more detectable components, comprising a scaffold,comprising one or more signal generating moieties, conjugated to anoligonucleotide sequence complementary to the oligonucleotide sequenceof the molecular probe, comprising: a) introducing a modified scaffold,comprising the one or more signal generating moieties, into a bufferedsolution; b) conjugating the modified scaffold with at least onemodified complementary oligonucleotide at greater than 90% efficiency toform scaffold-complementary oligonucleotide conjugates; and c) isolatingthe scaffold-complementary oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and iii)providing the one or more molecular probes to the complex samplecomprising the one or more biological targets; iv) specifically bindingthe one or more biological targets with the binding moieties of the oneor more molecular probes; v) providing the one or more detectablecomponents to the complex sample; vi) hydridizing the oligonucleotidesequences of the one or more target-bound molecular probes to thecomplementary oligonucleotide sequences of the one or more detectablecomponents; and vii) detecting one or more signals generated from theone or more hydridized detectable components; wherein: a) theconjugation between the oligonucleotide sequence and the binding moietyand conjugation between the complementary oligonucleotide sequence andthe scaffold, comprises a covalent bond linkage, comprising a hydrazone,oxime, triazine, or other bond, wherein the formation of the conjugatesare at least 90% efficient; b) the binding moiety comprises a bindingaffinity of less than 10⁻⁴ M for the one or more biological targets; c)the hydridization efficiency of the oligonucleotide sequence to thecomplementary oligonucleotide sequence is at least 50%; d) the one ormore molecular probes have substantially the same solubility as the oneor more binding moieties, respectively; e) the one or more molecularprobes comprise molecular weights of between about 15,000 Daltons toabout 450,000 Daltons; f) the method of detection generates less falsepositives than secondary antibody detection methods; g) the prepared andisolated one or more molecular probes and one or more detectablecomponents have at least 90% purity; h) the solubility of the one ormore molecular probes minimizes non-specific binding to the one or morebiological targets; i) the sample is homogeneous or heterogenousmixture; j) the sample comprises biological fluids or fluidizedbiological tissue; k) the sample comprises cells, membranes, biologicalmolecules, metabolites, or disease biomarkers; l) the sample comprises arange of analytes having a wide range of binding specificities; m) thetime of conducting the method, from the start of preparation to the endof detection is between about 2-3 hours; n) the one or more molecularprobes are neutral in charge; o) the scaffold comprises a dendrimer,polysaccharide, or a dextran; and p) the one or more molecular probescomprise a range of specificities for the one or more biologicaltargets.

Example 71A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) preparing and isolating oneor more molecular probes, comprising a binding moiety conjugated to anoligonucleotide sequence, comprising: a) introducing a modified bindingmoiety into a buffered solution; b) conjugating the modified bindingmoieties with at least one modified oligonucleotide at greater than 90%efficiency to form binding moiety-oligonucleotide conjugates; and c)isolating the binding moiety-oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and ii) preparingand isolating one or more detectable components, comprising a scaffold,comprising one or more signal generating moieties, conjugated to anoligonucleotide sequence complementary to the oligonucleotide sequenceof the molecular probe, comprising: a) introducing a modified scaffold,comprising the one or more signal generating moieties, into a bufferedsolution; b) conjugating the modified scaffold with at least onemodified complementary oligonucleotide at greater than 90% efficiency toform scaffold-complementary oligonucleotide conjugates; and c) isolatingthe scaffold-complementary oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and iii)providing the one or more molecular probes to the complex samplecomprising the one or more biological targets; iv) specifically bindingthe one or more biological targets with the binding moieties of the oneor more molecular probes; v) providing the one or more detectablecomponents to the complex sample; vi) hydridizing the oligonucleotidesequences of the one or more target-bound molecular probes to thecomplementary oligonucleotide sequences of the one or more detectablecomponents; and vii) detecting one or more signals generated from theone or more hydridized detectable components; wherein: a) theconjugation between the oligonucleotide sequence and the binding moietyand conjugation between the complementary oligonucleotide sequence andthe scaffold, comprises a covalent bond linkage, comprising a hydrazone,oxime, triazine, or other bond, wherein the formation of the conjugatesare at least 90% efficient; b) the binding moiety comprises a bindingaffinity of less than 10⁻⁴ M for the one or more biological targets; c)the hydridization efficiency of the oligonucleotide sequence to thecomplementary oligonucleotide sequence is at least 50%; d) the one ormore molecular probes have substantially the same solubility as the oneor more binding moieties, respectively; e) the one or more molecularprobes comprise molecular weights of between about 15,000 Daltons toabout 450,000 Daltons; f) the method of detection generates less falsepositives than secondary antibody detection methods; g) the prepared andisolated one or more molecular probes and one or more detectablecomponents have at least 90% purity; h) the solubility of the one ormore molecular probes minimizes non-specific binding to the one or morebiological targets; i) the sample is homogeneous or heterogenousmixture; j) the sample comprises biological fluids or fluidizedbiological tissue; k) the sample comprises cells, membranes, biologicalmolecules, metabolites, or disease biomarkers; l) the sample comprises arange of analytes having a wide range of binding specificities; m) thetime of conducting the method, from the start of preparation to the endof detection is between about 2-3 hours; n) the one or more molecularprobes are neutral in charge; o) the scaffold comprises a dendrimer,polysaccharide, or a dextran; p) the one or more molecular probescomprise a range of specificities for the one or more biologicaltargets; and q) the detection assay comprises a singleplex or multiplexdetection assay, comprising immunodetection, flow cytometry,immunohistochemistry, microspcopy, imaging, high content screening(HCS), ELISA, ELISpot, arrays, bead arrays, or combinations orderivatives thereof.

Example 72A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) preparing and isolating oneor more molecular probes, comprising a binding moiety conjugated to anoligonucleotide sequence, comprising: a) introducing a modified bindingmoiety into a buffered solution; b) conjugating the modified bindingmoieties with at least one modified oligonucleotide at greater than 90%efficiency to form binding moiety-oligonucleotide conjugates; and c)isolating the binding moiety-oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and ii) preparingand isolating one or more detectable components, comprising a scaffold,comprising one or more signal generating moieties, conjugated to anoligonucleotide sequence complementary to the oligonucleotide sequenceof the molecular probe, comprising: a) introducing a modified scaffold,comprising the one or more signal generating moieties, into a bufferedsolution; b) conjugating the modified scaffold with at least onemodified complementary oligonucleotide at greater than 90% efficiency toform scaffold-complementary oligonucleotide conjugates; and c) isolatingthe scaffold-complementary oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and iii)providing the one or more molecular probes to the complex samplecomprising the one or more biological targets; iv) specifically bindingthe one or more biological targets with the binding moieties of the oneor more molecular probes; v) providing the one or more detectablecomponents to the complex sample; vi) hydridizing the oligonucleotidesequences of the one or more target-bound molecular probes to thecomplementary oligonucleotide sequences of the one or more detectablecomponents; and vii) detecting one or more signals generated from theone or more hydridized detectable components; wherein: a) theconjugation between the oligonucleotide sequence and the binding moietyand conjugation between the complementary oligonucleotide sequence andthe scaffold, comprises a covalent bond linkage, comprising a hydrazone,oxime, triazine, or other bond, wherein the formation of the conjugatesare at least 90% efficient; b) the binding moiety comprises a bindingaffinity of less than 10⁻⁴ M for the one or more biological targets; c)the hydridization efficiency of the oligonucleotide sequence to thecomplementary oligonucleotide sequence is at least 50%; d) the one ormore molecular probes have substantially the same solubility as the oneor more binding moieties, respectively; e) the one or more molecularprobes comprise molecular weights of between about 15,000 Daltons toabout 450,000 Daltons; f) the method of detection generates less falsepositives than secondary antibody detection methods; g) the prepared andisolated one or more molecular probes and one or more detectablecomponents have at least 90% purity; h) the solubility of the one ormore molecular probes minimizes non-specific binding to the one or morebiological targets; i) the sample is homogeneous or heterogenousmixture; j) the sample comprises biological fluids or fluidizedbiological tissue; k) the sample comprises cells, membranes, biologicalmolecules, metabolites, or disease biomarkers; l) the sample comprises arange of analytes having a wide range of binding specificities; m) thetime of conducting the method, from the start of preparation to the endof detection is between about 2-3 hours; n) the one or more molecularprobes are neutral in charge; o) the scaffold comprises a dendrimer,polysaccharide, or a dextran; p) the one or more molecular probescomprise a range of specificities for the one or more biologicaltargets; q) the detection assay comprises a singleplex or multiplexdetection assay, comprising immunodetection, flow cytometry,immunohistochemistry, microspcopy, imaging, high content screening(HCS), ELISA, ELISpot, arrays, bead arrays, or combinations orderivatives thereof; r) the binding moiety comprises an antibody, aprotein, a peptide, a carbohydrate, a nuclear receptor, a smallmolecule, or combinations or derivatives thereof.

Example 73A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) preparing and isolating oneor more molecular probes, comprising a binding moiety conjugated to anoligonucleotide sequence, comprising: a) introducing a modified bindingmoiety into a buffered solution; b) conjugating the modified bindingmoieties with at least one modified oligonucleotide at greater than 90%efficiency to form binding moiety-oligonucleotide conjugates; and c)isolating the binding moiety-oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and ii) preparingand isolating one or more detectable components, comprising a scaffold,comprising one or more signal generating moieties, conjugated to anoligonucleotide sequence complementary to the oligonucleotide sequenceof the molecular probe, comprising: a) introducing a modified scaffold,comprising the one or more signal generating moieties, into a bufferedsolution; b) conjugating the modified scaffold with at least onemodified complementary oligonucleotide at greater than 90% efficiency toform scaffold-complementary oligonucleotide conjugates; and c) isolatingthe scaffold-complementary oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and iii)providing the one or more molecular probes to the complex samplecomprising the one or more biological targets; iv) specifically bindingthe one or more biological targets with the binding moieties of the oneor more molecular probes; v) providing the one or more detectablecomponents to the complex sample; vi) hydridizing the oligonucleotidesequences of the one or more target-bound molecular probes to thecomplementary oligonucleotide sequences of the one or more detectablecomponents; and vii) detecting one or more signals generated from theone or more hydridized detectable components; wherein: a) theconjugation between the oligonucleotide sequence and the binding moietyand conjugation between the complementary oligonucleotide sequence andthe scaffold, comprises a covalent bond linkage, comprising a hydrazone,oxime, triazine, or other bond, wherein the formation of the conjugatesare at least 90% efficient; b) the binding moiety comprises a bindingaffinity of less than 10⁻⁴ M for the one or more biological targets; c)the hydridization efficiency of the oligonucleotide sequence to thecomplementary oligonucleotide sequence is at least 50%; d) the one ormore molecular probes have substantially the same solubility as the oneor more binding moieties, respectively; e) the one or more molecularprobes comprise molecular weights of between about 15,000 Daltons toabout 450,000 Daltons; f) the method of detection generates less falsepositives than secondary antibody detection methods; g) the prepared andisolated one or more molecular probes and one or more detectablecomponents have at least 90% purity; h) the solubility of the one ormore molecular probes minimizes non-specific binding to the one or morebiological targets; i) the sample is homogeneous or heterogenousmixture; j) the sample comprises biological fluids or fluidizedbiological tissue; k) the sample comprises cells, membranes, biologicalmolecules, metabolites, or disease biomarkers; l) the sample comprises arange of analytes having a wide range of binding specificities; m) thetime of conducting the method, from the start of preparation to the endof detection is between about 2-3 hours; n) the one or more molecularprobes are neutral in charge; o) the scaffold comprises a dendrimer,polysaccharide, or a dextran; p) the one or more molecular probescomprise a range of specificities for the one or more biologicaltargets; q) the detection assay comprises a singleplex or multiplexdetection assay, comprising immunodetection, flow cytometry,immunohistochemistry, microspcopy, imaging, high content screening(HCS), ELISA, ELISpot, arrays, bead arrays, or combinations orderivatives thereof; r) the binding moiety comprises an antibody, aprotein, a peptide, a carbohydrate, a nuclear receptor, a smallmolecule, or combinations or derivatives thereof; and s) the one or morebiological target comprises an antigen, a pathogen, a protein, apeptide, an epitope, a carbohydrate-containing molecule, a smallmolecule, or combinations or derivatives thereof.

Example 74A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) preparing and isolating oneor more molecular probes, comprising a binding moiety conjugated to anoligonucleotide sequence, comprising: a) introducing a modified bindingmoiety into a buffered solution; b) conjugating the modified bindingmoieties with at least one modified oligonucleotide at greater than 90%efficiency to form binding moiety-oligonucleotide conjugates; and c)isolating the binding moiety-oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and ii) preparingand isolating one or more detectable components, comprising a scaffold,comprising one or more signal generating moieties, conjugated to anoligonucleotide sequence complementary to the oligonucleotide sequenceof the molecular probe, comprising: a) introducing a modified scaffold,comprising the one or more signal generating moieties, into a bufferedsolution; b) conjugating the modified scaffold with at least onemodified complementary oligonucleotide at greater than 90% efficiency toform scaffold-complementary oligonucleotide conjugates; and c) isolatingthe scaffold-complementary oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and iii)providing the one or more molecular probes to the complex samplecomprising the one or more biological targets; iv) specifically bindingthe one or more biological targets with the binding moieties of the oneor more molecular probes; v) providing the one or more detectablecomponents to the complex sample; vi) hydridizing the oligonucleotidesequences of the one or more target-bound molecular probes to thecomplementary oligonucleotide sequences of the one or more detectablecomponents; and vii) detecting one or more signals generated from theone or more hydridized detectable components; wherein: a) theconjugation between the oligonucleotide sequence and the binding moietyand conjugation between the complementary oligonucleotide sequence andthe scaffold, comprises a covalent bond linkage, comprising a hydrazone,oxime, triazine, or other bond, wherein the formation of the conjugatesare at least 90% efficient; b) the binding moiety comprises a bindingaffinity of less than 10⁻⁴ M for the one or more biological targets; c)the hydridization efficiency of the oligonucleotide sequence to thecomplementary oligonucleotide sequence is at least 50%; d) the one ormore molecular probes have substantially the same solubility as the oneor more binding moieties, respectively; e) the one or more molecularprobes comprise molecular weights of between about 15,000 Daltons toabout 450,000 Daltons; f) the method of detection generates less falsepositives than secondary antibody detection methods; g) the prepared andisolated one or more molecular probes and one or more detectablecomponents have at least 90% purity; h) the solubility of the one ormore molecular probes minimizes non-specific binding to the one or morebiological targets; i) the sample is homogeneous or heterogenousmixture; j) the sample comprises biological fluids or fluidizedbiological tissue; k) the sample comprises cells, membranes, biologicalmolecules, metabolites, or disease biomarkers; l) the sample comprises arange of analytes having a wide range of binding specificities; m) thetime of conducting the method, from the start of preparation to the endof detection is between about 2-3 hours; n) the one or more molecularprobes are neutral in charge; o) the scaffold comprises a dendrimer,polysaccharide, or a dextran; p) the one or more molecular probescomprise a range of specificities for the one or more biologicaltargets; q) the detection assay comprises a singleplex or multiplexdetection assay, comprising immunodetection, flow cytometry,immunohistochemistry, microspcopy, imaging, high content screening(HCS), ELISA, ELISpot, arrays, bead arrays, or combinations orderivatives thereof; r) the binding moiety comprises an antibody, aprotein, a peptide, a carbohydrate, a nuclear receptor, a smallmolecule, or combinations or derivatives thereof; s) the one or morebiological target comprises an antigen, a pathogen, a protein, apeptide, an epitope, a carbohydrate-containing molecule, a smallmolecule, or combinations or derivatives thereof; and t) the one or moresignal generating moieties, comprise one or more fluorophors,biofluorescent proteins, quantum dots, Raman particles, or combinationsor derivatives thereof.

Example 75A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) preparing and isolating oneor more molecular probes, comprising a binding moiety conjugated to anoligonucleotide sequence, comprising: a) introducing a modified bindingmoiety into a buffered solution; b) conjugating the modified bindingmoieties with at least one modified oligonucleotide at greater than 90%efficiency to form binding moiety-oligonucleotide conjugates; and c)isolating the binding moiety-oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and ii) preparingand isolating one or more detectable components, comprising a scaffold,comprising one or more signal generating moieties, conjugated to anoligonucleotide sequence complementary to the oligonucleotide sequenceof the molecular probe, comprising: a) introducing a modified scaffold,comprising the one or more signal generating moieties, into a bufferedsolution; b) conjugating the modified scaffold with at least onemodified complementary oligonucleotide at greater than 90% efficiency toform scaffold-complementary oligonucleotide conjugates; and c) isolatingthe scaffold-complementary oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and iii)providing the one or more molecular probes to the complex samplecomprising the one or more biological targets; iv) specifically bindingthe one or more biological targets with the binding moieties of the oneor more molecular probes; v) providing the one or more detectablecomponents to the complex sample; vi) hydridizing the oligonucleotidesequences of the one or more target-bound molecular probes to thecomplementary oligonucleotide sequences of the one or more detectablecomponents; and vii) detecting one or more signals generated from theone or more hydridized detectable components; wherein: a) theconjugation between the oligonucleotide sequence and the binding moietyand conjugation between the complementary oligonucleotide sequence andthe scaffold, comprises a covalent bond linkage, comprising a hydrazone,oxime, triazine, or other bond, wherein the formation of the conjugatesare at least 90% efficient; b) the binding moiety comprises a bindingaffinity of less than 10⁻⁴ M for the one or more biological targets; c)the hydridization efficiency of the oligonucleotide sequence to thecomplementary oligonucleotide sequence is at least 50%; d) the one ormore molecular probes have substantially the same solubility as the oneor more binding moieties, respectively; e) the one or more molecularprobes comprise molecular weights of between about 15,000 Daltons toabout 450,000 Daltons; f) the method of detection generates less falsepositives than secondary antibody detection methods; g) the prepared andisolated one or more molecular probes and one or more detectablecomponents have at least 90% purity; h) the solubility of the one ormore molecular probes minimizes non-specific binding to the one or morebiological targets; i) the sample is homogeneous or heterogenousmixture; j) the sample comprises biological fluids or fluidizedbiological tissue; k) the sample comprises cells, membranes, biologicalmolecules, metabolites, or disease biomarkers; l) the sample comprises arange of analytes having a wide range of binding specificities; m) thetime of conducting the method, from the start of preparation to the endof detection is between about 2-3 hours; n) the one or more molecularprobes are neutral in charge; o) the scaffold comprises a dendrimer,polysaccharide, or a dextran; p) the one or more molecular probescomprise a range of specificities for the one or more biologicaltargets; q) the detection assay comprises a singleplex or multiplexdetection assay, comprising immunodetection, flow cytometry,immunohistochemistry, microspcopy, imaging, high content screening(HCS), ELISA, ELISpot, arrays, bead arrays, or combinations orderivatives thereof; r) the binding moiety comprises an antibody, aprotein, a peptide, a carbohydrate, a nuclear receptor, a smallmolecule, or combinations or derivatives thereof; s) the one or morebiological target comprises an antigen, a pathogen, a protein, apeptide, an epitope, a carbohydrate-containing molecule, a smallmolecule, or combinations or derivatives thereof; t) the one or moresignal generating moieties, comprise one or more fluorophors,biofluorescent proteins, quantum dots, Raman particles, or combinationsor derivatives thereof; and u) the one or more signal generatingmoieties provides an enhanced signal that minimizes detection errorsfrom background noise.

Example 76A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) preparing and isolating oneor more molecular probes, comprising a binding moiety conjugated to anoligonucleotide sequence, comprising: a) introducing a modified bindingmoiety into a buffered solution; b) conjugating the modified bindingmoieties with at least one modified oligonucleotide at greater than 90%efficiency to form binding moiety-oligonucleotide conjugates; and c)isolating the binding moiety-oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and ii) preparingand isolating one or more detectable components, comprising a scaffold,comprising one or more signal generating moieties, conjugated to anoligonucleotide sequence complementary to the oligonucleotide sequenceof the molecular probe, comprising: a) introducing a modified scaffold,comprising the one or more signal generating moieties, into a bufferedsolution; b) conjugating the modified scaffold with at least onemodified complementary oligonucleotide at greater than 90% efficiency toform scaffold-complementary oligonucleotide conjugates; and c) isolatingthe scaffold-complementary oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and iii)providing the one or more molecular probes to the complex samplecomprising the one or more biological targets; iv) specifically bindingthe one or more biological targets with the binding moieties of the oneor more molecular probes; v) providing the one or more detectablecomponents to the complex sample; vi) hydridizing the oligonucleotidesequences of the one or more target-bound molecular probes to thecomplementary oligonucleotide sequences of the one or more detectablecomponents; and vii) detecting one or more signals generated from theone or more hydridized detectable components; wherein: a) theconjugation between the oligonucleotide sequence and the binding moietyand conjugation between the complementary oligonucleotide sequence andthe scaffold, comprises a covalent bond linkage, comprising a hydrazone,oxime, triazine, or other bond, wherein the formation of the conjugatesare at least 90% efficient; b) the binding moiety comprises a bindingaffinity of less than 10⁻⁴ M for the one or more biological targets; c)the hydridization efficiency of the oligonucleotide sequence to thecomplementary oligonucleotide sequence is at least 50%; d) the one ormore molecular probes have substantially the same solubility as the oneor more binding moieties, respectively; e) the one or more molecularprobes comprise molecular weights of between about 15,000 Daltons toabout 450,000 Daltons; f) the method of detection generates less falsepositives than secondary antibody detection methods; g) the prepared andisolated one or more molecular probes and one or more detectablecomponents have at least 90% purity; h) the solubility of the one ormore molecular probes minimizes non-specific binding to the one or morebiological targets; i) the sample is homogeneous or heterogenousmixture; j) the sample comprises biological fluids or fluidizedbiological tissue; k) the sample comprises cells, membranes, biologicalmolecules, metabolites, or disease biomarkers; l) the sample comprises arange of analytes having a wide range of binding specificities; m) thetime of conducting the method, from the start of preparation to the endof detection is between about 2-3 hours; n) the one or more molecularprobes are neutral in charge; o) the scaffold comprises a dendrimer,polysaccharide, or a dextran; p) the one or more molecular probescomprise a range of specificities for the one or more biologicaltargets; q) the detection assay comprises a singleplex or multiplexdetection assay, comprising immunodetection, flow cytometry,immunohistochemistry, microspcopy, imaging, high content screening(HCS), ELISA, ELISpot, arrays, bead arrays, or combinations orderivatives thereof; r) the binding moiety comprises an antibody, aprotein, a peptide, a carbohydrate, a nuclear receptor, a smallmolecule, or combinations or derivatives thereof; s) the one or morebiological target comprises an antigen, a pathogen, a protein, apeptide, an epitope, a carbohydrate-containing molecule, a smallmolecule, or combinations or derivatives thereof; t) the one or moresignal generating moieties, comprise one or more fluorophors,biofluorescent proteins, quantum dots, Raman particles, or combinationsor derivatives thereof; u) the one or more signal generating moietiesprovides an enhanced signal that minimizes detection errors frombackground noise; and v) the one or more molecular probes and/or the oneor more detectable components further comprise a spacer group,comprising a polymerized ethylene oxide, a PEG, or a PEO.

Example 77A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) preparing and isolating oneor more molecular probes, comprising a binding moiety conjugated to anoligonucleotide sequence, comprising: a) introducing a modified bindingmoiety into a buffered solution; b) conjugating the modified bindingmoieties with at least one modified oligonucleotide at greater than 90%efficiency to form binding moiety-oligonucleotide conjugates; and c)isolating the binding moiety-oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and ii) preparingand isolating one or more detectable components, comprising a scaffold,comprising one or more signal generating moieties, conjugated to anoligonucleotide sequence complementary to the oligonucleotide sequenceof the molecular probe, comprising: a) introducing a modified scaffold,comprising the one or more signal generating moieties, into a bufferedsolution; b) conjugating the modified scaffold with at least onemodified complementary oligonucleotide at greater than 90% efficiency toform scaffold-complementary oligonucleotide conjugates; and c) isolatingthe scaffold-complementary oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and iii)providing the one or more molecular probes to the complex samplecomprising the one or more biological targets; iv) specifically bindingthe one or more biological targets with the binding moieties of the oneor more molecular probes; v) providing the one or more detectablecomponents to the complex sample; vi) hydridizing the oligonucleotidesequences of the one or more target-bound molecular probes to thecomplementary oligonucleotide sequences of the one or more detectablecomponents; and vii) detecting one or more signals generated from theone or more hydridized detectable components; wherein: a) theconjugation between the oligonucleotide sequence and the binding moietyand conjugation between the complementary oligonucleotide sequence andthe scaffold, comprises a covalent bond linkage, comprising a hydrazone,oxime, triazine, or other bond, wherein the formation of the conjugatesare at least 90% efficient; b) the binding moiety comprises a bindingaffinity of less than 10⁻⁴ M for the one or more biological targets; c)the hydridization efficiency of the oligonucleotide sequence to thecomplementary oligonucleotide sequence is at least 50%; d) the one ormore molecular probes have substantially the same solubility as the oneor more binding moieties, respectively; e) the one or more molecularprobes comprise molecular weights of between about 15,000 Daltons toabout 450,000 Daltons; f) the method of detection generates less falsepositives than secondary antibody detection methods; g) the prepared andisolated one or more molecular probes and one or more detectablecomponents have at least 90% purity; h) the solubility of the one ormore molecular probes minimizes non-specific binding to the one or morebiological targets; i) the sample is homogeneous or heterogenousmixture; j) the sample comprises biological fluids or fluidizedbiological tissue; k) the sample comprises cells, membranes, biologicalmolecules, metabolites, or disease biomarkers; l) the sample comprises arange of analytes having a wide range of binding specificities; m) thetime of conducting the method, from the start of preparation to the endof detection is between about 2-3 hours; n) the one or more molecularprobes are neutral in charge; o) the scaffold comprises a dendrimer,polysaccharide, or a dextran; p) the one or more molecular probescomprise a range of specificities for the one or more biologicaltargets; q) the detection assay comprises a singleplex or multiplexdetection assay, comprising immunodetection, flow cytometry,immunohistochemistry, microspcopy, imaging, high content screening(HCS), ELISA, ELISpot, arrays, bead arrays, or combinations orderivatives thereof; r) the binding moiety comprises an antibody, aprotein, a peptide, a carbohydrate, a nuclear receptor, a smallmolecule, or combinations or derivatives thereof; s) the one or morebiological target comprises an antigen, a pathogen, a protein, apeptide, an epitope, a carbohydrate-containing molecule, a smallmolecule, or combinations or derivatives thereof; t) the one or moresignal generating moieties, comprise one or more fluorophors,biofluorescent proteins, quantum dots, Raman particles, or combinationsor derivatives thereof; u) the one or more signal generating moietiesprovides an enhanced signal that minimizes detection errors frombackground noise; v) the one or more molecular probes and/or the one ormore detectable components further comprise a spacer group, comprising apolymerized ethylene oxide, a PEG, or a PEO; and w) the modified bindingmoiety, the modified scaffold, the oligonucleotide sequence, and/or thecomplementary oligonucleotide sequence comprise HyNic or 4-FB.

Example 78A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) preparing and isolating oneor more molecular probes, comprising a binding moiety conjugated to anoligonucleotide sequence, comprising: a) introducing a modified bindingmoiety into a buffered solution; b) conjugating the modified bindingmoieties with at least one modified oligonucleotide at greater than 90%efficiency to form binding moiety-oligonucleotide conjugates; and c)isolating the binding moiety-oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and ii) preparingand isolating one or more detectable components, comprising a scaffold,comprising one or more signal generating moieties, conjugated to anoligonucleotide sequence complementary to the oligonucleotide sequenceof the molecular probe, comprising: a) introducing a modified scaffold,comprising the one or more signal generating moieties, into a bufferedsolution; b) conjugating the modified scaffold with at least onemodified complementary oligonucleotide at greater than 90% efficiency toform scaffold-complementary oligonucleotide conjugates; and c) isolatingthe scaffold-complementary oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and iii)providing the one or more molecular probes to the complex samplecomprising the one or more biological targets; iv) specifically bindingthe one or more biological targets with the binding moieties of the oneor more molecular probes; v) providing the one or more detectablecomponents to the complex sample; vi) hydridizing the oligonucleotidesequences of the one or more target-bound molecular probes to thecomplementary oligonucleotide sequences of the one or more detectablecomponents; and vii) detecting one or more signals generated from theone or more hydridized detectable components; wherein: a) theconjugation between the oligonucleotide sequence and the binding moietyand conjugation between the complementary oligonucleotide sequence andthe scaffold, comprises a covalent bond linkage, comprising a hydrazone,oxime, triazine, or other bond, wherein the formation of the conjugatesare at least 90% efficient; b) the binding moiety comprises a bindingaffinity of less than 10⁻⁴ M for the one or more biological targets; c)the hydridization efficiency of the oligonucleotide sequence to thecomplementary oligonucleotide sequence is at least 50%; d) the one ormore molecular probes have substantially the same solubility as the oneor more binding moieties, respectively; e) the one or more molecularprobes comprise molecular weights of between about 15,000 Daltons toabout 450,000 Daltons; f) the method of detection generates less falsepositives than secondary antibody detection methods; g) the prepared andisolated one or more molecular probes and one or more detectablecomponents have at least 90% purity; h) the solubility of the one ormore molecular probes minimizes non-specific binding to the one or morebiological targets; i) the sample is homogeneous or heterogenousmixture; j) the sample comprises biological fluids or fluidizedbiological tissue; k) the sample comprises cells, membranes, biologicalmolecules, metabolites, or disease biomarkers; l) the sample comprises arange of analytes having a wide range of binding specificities; m) thetime of conducting the method, from the start of preparation to the endof detection is between about 2-3 hours; n) the one or more molecularprobes are neutral in charge; o) the scaffold comprises a dendrimer,polysaccharide, or a dextran; p) the one or more molecular probescomprise a range of specificities for the one or more biologicaltargets; q) the detection assay comprises a singleplex or multiplexdetection assay, comprising immunodetection, flow cytometry,immunohistochemistry, microspcopy, imaging, high content screening(HCS), ELISA, ELISpot, arrays, bead arrays, or combinations orderivatives thereof; r) the binding moiety comprises an antibody, aprotein, a peptide, a carbohydrate, a nuclear receptor, a smallmolecule, or combinations or derivatives thereof; s) the one or morebiological target comprises an antigen, a pathogen, a protein, apeptide, an epitope, a carbohydrate-containing molecule, a smallmolecule, or combinations or derivatives thereof; t) the one or moresignal generating moieties, comprise one or more fluorophors,biofluorescent proteins, quantum dots, Raman particles, or combinationsor derivatives thereof; u) the one or more signal generating moietiesprovides an enhanced signal that minimizes detection errors frombackground noise; v) the one or more molecular probes and/or the one ormore detectable components further comprise a spacer group, comprising apolymerized ethylene oxide, a PEG, or a PEO; w) the modified bindingmoiety, the modified scaffold, the oligonucleotide sequence, and/or thecomplementary oligonucleotide sequence comprise HyNic or 4-FB; and x)the one or more molecular probes comprise unique, distinguishable,and/or specifically designed oligonucleotide sequences.

Example 79A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) preparing and isolating oneor more molecular probes, comprising a binding moiety conjugated to anoligonucleotide sequence, comprising: a) introducing a modified bindingmoiety into a buffered solution; b) conjugating the modified bindingmoieties with at least one modified oligonucleotide at greater than 90%efficiency to form binding moiety-oligonucleotide conjugates; and c)isolating the binding moiety-oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and ii) preparingand isolating one or more detectable components, comprising a scaffold,comprising one or more signal generating moieties, conjugated to anoligonucleotide sequence complementary to the oligonucleotide sequenceof the molecular probe, comprising: a) introducing a modified scaffold,comprising the one or more signal generating moieties, into a bufferedsolution; b) conjugating the modified scaffold with at least onemodified complementary oligonucleotide at greater than 90% efficiency toform scaffold-complementary oligonucleotide conjugates; and c) isolatingthe scaffold-complementary oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and iii)providing the one or more molecular probes to the complex samplecomprising the one or more biological targets; iv) specifically bindingthe one or more biological targets with the binding moieties of the oneor more molecular probes; v) providing the one or more detectablecomponents to the complex sample; vi) hydridizing the oligonucleotidesequences of the one or more target-bound molecular probes to thecomplementary oligonucleotide sequences of the one or more detectablecomponents; and vii) detecting one or more signals generated from theone or more hydridized detectable components; wherein: a) theconjugation between the oligonucleotide sequence and the binding moietyand conjugation between the complementary oligonucleotide sequence andthe scaffold, comprises a covalent bond linkage, comprising a hydrazone,oxime, triazine, or other bond, wherein the formation of the conjugatesare at least 90% efficient; b) the binding moiety comprises a bindingaffinity of less than 10⁻⁴ M for the one or more biological targets; c)the hydridization efficiency of the oligonucleotide sequence to thecomplementary oligonucleotide sequence is at least 50%; d) the one ormore molecular probes have substantially the same solubility as the oneor more binding moieties, respectively; e) the one or more molecularprobes comprise molecular weights of between about 15,000 Daltons toabout 450,000 Daltons; f) the method of detection generates less falsepositives than secondary antibody detection methods; g) the prepared andisolated one or more molecular probes and one or more detectablecomponents have at least 90% purity; h) the solubility of the one ormore molecular probes minimizes non-specific binding to the one or morebiological targets; i) the sample is homogeneous or heterogenousmixture; j) the sample comprises biological fluids or fluidizedbiological tissue; k) the sample comprises cells, membranes, biologicalmolecules, metabolites, or disease biomarkers; l) the sample comprises arange of analytes having a wide range of binding specificities; m) thetime of conducting the method, from the start of preparation to the endof detection is between about 2-3 hours; n) the one or more molecularprobes are neutral in charge; o) the scaffold comprises a dendrimer,polysaccharide, or a dextran; p) the one or more molecular probescomprise a range of specificities for the one or more biologicaltargets; q) the detection assay comprises a singleplex or multiplexdetection assay, comprising immunodetection, flow cytometry,immunohistochemistry, microspcopy, imaging, high content screening(HCS), ELISA, ELISpot, arrays, bead arrays, or combinations orderivatives thereof; r) the binding moiety comprises an antibody, aprotein, a peptide, a carbohydrate, a nuclear receptor, a smallmolecule, or combinations or derivatives thereof; s) the one or morebiological target comprises an antigen, a pathogen, a protein, apeptide, an epitope, a carbohydrate-containing molecule, a smallmolecule, or combinations or derivatives thereof; t) the one or moresignal generating moieties, comprise one or more fluorophors,biofluorescent proteins, quantum dots, Raman particles, or combinationsor derivatives thereof; u) the one or more signal generating moietiesprovides an enhanced signal that minimizes detection errors frombackground noise; v) the one or more molecular probes and/or the one ormore detectable components further comprise a spacer group, comprising apolymerized ethylene oxide, a PEG, or a PEO; w) the modified bindingmoiety, the modified scaffold, the oligonucleotide sequence, and/or thecomplementary oligonucleotide sequence comprise HyNic or 4-FB; x) theone or more molecular probes comprise unique, distinguishable, and/orspecifically designed oligonucleotide sequences; and y) the one or moredetectable components comprise unique, distinguishable, and/orspecifically designed complementary oligonucleotide sequences.

Example 80A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) preparing and isolating oneor more molecular probes, comprising a binding moiety conjugated to anoligonucleotide sequence, comprising: a) introducing a modified bindingmoiety into a buffered solution; b) conjugating the modified bindingmoieties with at least one modified oligonucleotide at greater than 90%efficiency to form binding moiety-oligonucleotide conjugates; and c)isolating the binding moiety-oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and ii) preparingand isolating one or more detectable components, comprising a scaffold,comprising one or more signal generating moieties, conjugated to anoligonucleotide sequence complementary to the oligonucleotide sequenceof the molecular probe, comprising: a) introducing a modified scaffold,comprising the one or more signal generating moieties, into a bufferedsolution; b) conjugating the modified scaffold with at least onemodified complementary oligonucleotide at greater than 90% efficiency toform scaffold-complementary oligonucleotide conjugates; and c) isolatingthe scaffold-complementary oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and iii)providing the one or more molecular probes to the complex samplecomprising the one or more biological targets; iv) specifically bindingthe one or more biological targets with the binding moieties of the oneor more molecular probes; v) providing the one or more detectablecomponents to the complex sample; vi) hydridizing the oligonucleotidesequences of the one or more target-bound molecular probes to thecomplementary oligonucleotide sequences of the one or more detectablecomponents; and vii) detecting one or more signals generated from theone or more hydridized detectable components; wherein: a) theconjugation between the oligonucleotide sequence and the binding moietyand conjugation between the complementary oligonucleotide sequence andthe scaffold, comprises a covalent bond linkage, comprising a hydrazone,oxime, triazine, or other bond, wherein the formation of the conjugatesare at least 90% efficient; b) the binding moiety comprises a bindingaffinity of less than 10⁻⁴ M for the one or more biological targets; c)the hydridization efficiency of the oligonucleotide sequence to thecomplementary oligonucleotide sequence is at least 50%; d) the one ormore molecular probes have substantially the same solubility as the oneor more binding moieties, respectively; e) the one or more molecularprobes comprise molecular weights of between about 15,000 Daltons toabout 450,000 Daltons; f) the method of detection generates less falsepositives than secondary antibody detection methods; g) the prepared andisolated one or more molecular probes and one or more detectablecomponents have at least 90% purity; h) the solubility of the one ormore molecular probes minimizes non-specific binding to the one or morebiological targets; i) the sample is homogeneous or heterogenousmixture; j) the sample comprises biological fluids or fluidizedbiological tissue; k) the sample comprises cells, membranes, biologicalmolecules, metabolites, or disease biomarkers; l) the sample comprises arange of analytes having a wide range of binding specificities; m) thetime of conducting the method, from the start of preparation to the endof detection is between about 2-3 hours; n) the one or more molecularprobes are neutral in charge; o) the scaffold comprises a dendrimer,polysaccharide, or a dextran; p) the one or more molecular probescomprise a range of specificities for the one or more biologicaltargets; q) the detection assay comprises a singleplex or multiplexdetection assay, comprising immunodetection, flow cytometry,immunohistochemistry, microspcopy, imaging, high content screening(HCS), ELISA, ELISpot, arrays, bead arrays, or combinations orderivatives thereof; r) the binding moiety comprises an antibody, aprotein, a peptide, a carbohydrate, a nuclear receptor, a smallmolecule, or combinations or derivatives thereof; s) the one or morebiological target comprises an antigen, a pathogen, a protein, apeptide, an epitope, a carbohydrate-containing molecule, a smallmolecule, or combinations or derivatives thereof; t) the one or moresignal generating moieties, comprise one or more fluorophors,biofluorescent proteins, quantum dots, Raman particles, or combinationsor derivatives thereof; u) the one or more signal generating moietiesprovides an enhanced signal that minimizes detection errors frombackground noise; v) the one or more molecular probes and/or the one ormore detectable components further comprise a spacer group, comprising apolymerized ethylene oxide, a PEG, or a PEO; w) the modified bindingmoiety, the modified scaffold, the oligonucleotide sequence, and/or thecomplementary oligonucleotide sequence comprise HyNic or 4-FB; x) theone or more molecular probes comprise unique, distinguishable, and/orspecifically designed oligonucleotide sequences; y) the one or moredetectable components comprise unique, distinguishable, and/orspecifically designed complementary oligonucleotide sequences; and z)the oligonucleotide sequences of the one or more molecular probes areuniquely and specifically designed to hybridize to the complementaryoligonucleotide sequence of the one or more detectable components.

Example 81A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) preparing and isolating oneor more molecular probes, comprising a binding moiety conjugated to anoligonucleotide sequence, comprising: a) introducing a modified bindingmoiety into a buffered solution; b) conjugating the modified bindingmoieties with at least one modified oligonucleotide at greater than 90%efficiency to form binding moiety-oligonucleotide conjugates; and c)isolating the binding moiety-oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and ii) preparingand isolating one or more detectable components, comprising a scaffold,comprising one or more signal generating moieties, conjugated to anoligonucleotide sequence complementary to the oligonucleotide sequenceof the molecular probe, comprising: a) introducing a modified scaffold,comprising the one or more signal generating moieties, into a bufferedsolution; b) conjugating the modified scaffold with at least onemodified complementary oligonucleotide at greater than 90% efficiency toform scaffold-complementary oligonucleotide conjugates; and c) isolatingthe scaffold-complementary oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and iii)providing the one or more molecular probes to the complex samplecomprising the one or more biological targets; iv) specifically bindingthe one or more biological targets with the binding moieties of the oneor more molecular probes; v) providing the one or more detectablecomponents to the complex sample; vi) hydridizing the oligonucleotidesequences of the one or more target-bound molecular probes to thecomplementary oligonucleotide sequences of the one or more detectablecomponents; and vii) detecting one or more signals generated from theone or more hydridized detectable components; wherein: a) theconjugation between the oligonucleotide sequence and the binding moietyand conjugation between the complementary oligonucleotide sequence andthe scaffold, comprises a covalent bond linkage, comprising a hydrazone,oxime, triazine, or other bond, wherein the formation of the conjugatesare at least 90% efficient; b) the binding moiety comprises a bindingaffinity of less than 10⁻⁴ M for the one or more biological targets; c)the hydridization efficiency of the oligonucleotide sequence to thecomplementary oligonucleotide sequence is at least 50%; d) the one ormore molecular probes have substantially the same solubility as the oneor more binding moieties, respectively; e) the one or more molecularprobes comprise molecular weights of between about 15,000 Daltons toabout 450,000 Daltons; f) the method of detection generates less falsepositives than secondary antibody detection methods; g) the prepared andisolated one or more molecular probes and one or more detectablecomponents have at least 90% purity; h) the solubility of the one ormore molecular probes minimizes non-specific binding to the one or morebiological targets; i) the sample is homogeneous or heterogenousmixture; j) the sample comprises biological fluids or fluidizedbiological tissue; k) the sample comprises cells, membranes, biologicalmolecules, metabolites, or disease biomarkers; l) the sample comprises arange of analytes having a wide range of binding specificities; m) thetime of conducting the method, from the start of preparation to the endof detection is between about 2-3 hours; n) the one or more molecularprobes are neutral in charge; o) the scaffold comprises a dendrimer,polysaccharide, or a dextran; p) the one or more molecular probescomprise a range of specificities for the one or more biologicaltargets; q) the detection assay comprises a singleplex or multiplexdetection assay, comprising immunodetection, flow cytometry,immunohistochemistry, microspcopy, imaging, high content screening(HCS), ELISA, ELISpot, arrays, bead arrays, or combinations orderivatives thereof; r) the binding moiety comprises an antibody, aprotein, a peptide, a carbohydrate, a nuclear receptor, a smallmolecule, or combinations or derivatives thereof; s) the one or morebiological target comprises an antigen, a pathogen, a protein, apeptide, an epitope, a carbohydrate-containing molecule, a smallmolecule, or combinations or derivatives thereof; t) the one or moresignal generating moieties, comprise one or more fluorophors,biofluorescent proteins, quantum dots, Raman particles, or combinationsor derivatives thereof; u) the one or more signal generating moietiesprovides an enhanced signal that minimizes detection errors frombackground noise; v) the one or more molecular probes and/or the one ormore detectable components further comprise a spacer group, comprising apolymerized ethylene oxide, a PEG, or a PEO; w) the modified bindingmoiety, the modified scaffold, the oligonucleotide sequence, and/or thecomplementary oligonucleotide sequence comprise HyNic or 4-FB; x) theone or more molecular probes comprise unique, distinguishable, and/orspecifically designed oligonucleotide sequences; y) the one or moredetectable components comprise unique, distinguishable, and/orspecifically designed complementary oligonucleotide sequences; z) theoligonucleotide sequences of the one or more molecular probes areuniquely and specifically designed to hybridize to the complementaryoligonucleotide sequence of the one or more detectable components; anda2) the oligonucleotide sequences and/or complementary oligonucleotidesequences, comprise 3′-oligonucleotides, 5′-oligonucleotides, LNAs,PNAs, or combinations or derivatives thereof.

Example 82A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) preparing and isolating oneor more molecular probes, comprising a binding moiety conjugated to anoligonucleotide sequence, comprising: a) introducing a modified bindingmoiety into a buffered solution; b) conjugating the modified bindingmoieties with at least one modified oligonucleotide at greater than 90%efficiency to form binding moiety-oligonucleotide conjugates; and c)isolating the binding moiety-oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and ii) preparingand isolating one or more detectable components, comprising a scaffold,comprising one or more signal generating moieties, conjugated to anoligonucleotide sequence, comprising: a) introducing a modifiedscaffold, comprising the one or more signal generating moieties, into abuffered solution; b) conjugating the modified scaffold with at leastone modified oligonucleotide at greater than 90% efficiency to formscaffold-oligonucleotide conjugates; and c) isolating thescaffold-oligonucleotide conjugates from the conjugation solution bybinding the conjugates to an immobilized binder, removing theunconjugated oligonucleotide in a wash step followed by release of thebound conjugate from the solid support; and iii) providing the one ormore molecular probes to the complex sample comprising the one or morebiological targets; iv) specifically binding the one or more biologicaltargets with the binding moieties of the one or more molecular probes;v) providing a universal adapter to the complex sample, wherein theuniversal adapter comprised an oligonucleotide sequence having a firstsequence segment complementary to the oligonucleotide sequence of themolecular probe and a second sequence segment complementary to theoligonucleotide sequence of the detectable component; vi) hydridizingthe oligonucleotide sequences of the one or more target-bound molecularprobes to the first oligonucleotide sequence segment of the universaladapter; vii) providing the one or more detectable components to thecomplex sample; viii) hydridizing the oligonucleotide sequences of theone or more detectable components to the second oligonucleotide sequencesegment of the universal adapter; and ix) detecting one or more signalsgenerated from the hydridized one or more detectable components;wherein: a) the conjugation between the oligonucleotide sequence and thebinding moiety and conjugation between the complementary oligonucleotidesequence and the scaffold, comprises a covalent bond linkage, comprisinga hydrazone, oxime, triazine, or other bond, wherein the formation ofthe conjugates are at least 90% efficient; b) the binding moietycomprises a binding affinity of less than 10⁻⁴ M for the one or morebiological targets; c) the hydridization efficiency of theoligonucleotide sequence to the complementary oligonucleotide sequenceis at least 50%; d) the one or more molecular probes have substantiallythe same solubility as the one or more binding moieties, respectively;e) the one or more molecular probes comprise molecular weights ofbetween about 15,000 Daltons to about 450,000 Daltons; f) the method ofdetection generates less false positives than secondary antibodydetection methods; g) the prepared and isolated one or more molecularprobes and one or more detectable components have at least 90% purity;h) the solubility of the one or more molecular probes minimizesnon-specific binding to the one or more biological targets; i) thesample is homogeneous or heterogenous mixture; j) the sample comprisesbiological fluids or fluidized biological tissue; k) the samplecomprises cells, membranes, biological molecules, metabolites, ordisease biomarkers; l) the sample comprises a range of analytes having awide range of binding specificities; m) the time of conducting themethod, from the start of preparation to the end of detection is betweenabout 2-3 hours; n) the one or more molecular probes are neutral incharge; o) the scaffold comprises a dendrimer, polysaccharide, or adextran; p) the one or more molecular probes comprise a range ofspecificities for the one or more biological targets; q) the detectionassay comprises a singleplex or multiplex detection assay, comprisingimmunodetection, flow cytometry, immunohistochemistry, microspcopy,imaging, high content screening (HCS), ELISA, ELISpot, arrays, beadarrays, or combinations or derivatives thereof; r) the binding moietycomprises an antibody, a protein, a peptide, a carbohydrate, a nuclearreceptor, a small molecule, or combinations or derivatives thereof; s)the one or more biological target comprises an antigen, a pathogen, aprotein, a peptide, an epitope, a carbohydrate-containing molecule, asmall molecule, or combinations or derivatives thereof; t) the one ormore signal generating moieties, comprise one or more fluorophors,biofluorescent proteins, quantum dots, Raman particles, or combinationsor derivatives thereof; u) the one or more signal generating moietiesprovides an enhanced signal that minimizes detection errors frombackground noise; v) the one or more molecular probes and/or the one ormore detectable components further comprise a spacer group, comprising apolymerized ethylene oxide, a PEG, or a PEO; w) the modified bindingmoiety, the modified scaffold, the oligonucleotide sequence, and/or thecomplementary oligonucleotide sequence comprise HyNic or 4-FB; x) theone or more molecular probes comprise unique, distinguishable, and/orspecifically designed oligonucleotide sequences; y) the one or moredetectable components comprise unique, distinguishable, and/orspecifically designed complementary oligonucleotide sequences; z) theoligonucleotide sequences of the one or more molecular probes areuniquely and specifically designed to hybridize to the complementaryoligonucleotide sequence of the one or more detectable components; anda2) the oligonucleotide sequences and/or complementary oligonucleotidesequences, comprise 3′-oligonucleotides, 5′-oligonucleotides, LNAs,PNAs, or combinations or derivatives thereof.

Example 83A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) preparing and isolating oneor more molecular probes, comprising a binding moiety conjugated to anoligonucleotide sequence, comprising: a) introducing a modified bindingmoiety into a buffered solution; b) conjugating the modified bindingmoieties with at least one modified oligonucleotide at greater than 90%efficiency to form binding moiety-oligonucleotide conjugates; and c)isolating the binding moiety-oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and ii) preparingand isolating one or more detectable components, comprising a scaffold,comprising one or more signal generating moieties, conjugated to anoligonucleotide sequence complementary to the oligonucleotide sequenceof the molecular probe, comprising: a) introducing a modified scaffold,comprising the one or more signal generating moieties, into a bufferedsolution; b) conjugating the modified scaffold with at least onemodified complementary oligonucleotide at greater than 90% efficiency toform scaffold-complementary oligonucleotide conjugates; and c) isolatingthe scaffold-complementary oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and iii)providing at least a first molecular probe and a second molecular probe,comprising a first binding moiety conjugated to a first oligonucleotidesequence and a second binding moiety conjugated to a secondoligonucleotide sequence, respectively, to the complex sample comprisingthe one or more biological targets; iv) specifically binding the one ormore biological targets with the binding moiety of the first molecularprobe and the binding moiety of the second molecular probe; v) providingthe one or more detectable components to the complex sample; vi)hydridizing the first oligonucleotide sequence of the first target-boundmolecular probe to a complementary oligonucleotide sequence conjugatedto a bead; vii) hydridizing the second oligonucleotide sequence of thesecond target-bound molecular probe to the complementary oligonucleotidesequences of the one or more detectable components; and viii) detectingone or more signals generated from the one or more hydridized detectablecomponents; wherein: a) the conjugation between the oligonucleotidesequence and the binding moiety and conjugation between thecomplementary oligonucleotide sequence and the scaffold, comprises acovalent bond linkage, comprising a hydrazone, oxime, triazine, or otherbond, wherein the formation of the conjugates are at least 90%efficient; b) the binding moiety comprises a binding affinity of lessthan 10⁻⁴ M for the one or more biological targets; c) the hydridizationefficiency of the oligonucleotide sequence to the complementaryoligonucleotide sequence is at least 50%; d) the one or more molecularprobes have substantially the same solubility as the one or more bindingmoieties, respectively; e) the one or more molecular probes comprisemolecular weights of between about 15,000 Daltons to about 450,000Daltons; f) the method of detection generates less false positives thansecondary antibody detection methods; g) the prepared and isolated oneor more molecular probes and one or more detectable components have atleast 90% purity; h) the solubility of the one or more molecular probesminimizes non-specific binding to the one or more biological targets; i)the sample is homogeneous or heterogenous mixture; j) the samplecomprises biological fluids or fluidized biological tissue; k) thesample comprises cells, membranes, biological molecules, metabolites, ordisease biomarkers; l) the sample comprises a range of analytes having awide range of binding specificities; m) the time of conducting themethod, from the start of preparation to the end of detection is betweenabout 2-3 hours; n) the one or more molecular probes are neutral incharge; o) the scaffold comprises a dendrimer, polysaccharide, or adextran; p) the one or more molecular probes comprise a range ofspecificities for the one or more biological targets; q) the detectionassay comprises a singleplex or multiplex detection assay, comprisingimmunodetection, flow cytometry, immunohistochemistry, microspcopy,imaging, high content screening (HCS), ELISA, ELISpot, arrays, beadarrays, or combinations or derivatives thereof; r) the binding moietycomprises an antibody, a protein, a peptide, a carbohydrate, a nuclearreceptor, a small molecule, or combinations or derivatives thereof; s)the one or more biological target comprises an antigen, a pathogen, aprotein, a peptide, an epitope, a carbohydrate-containing molecule, asmall molecule, or combinations or derivatives thereof; t) the one ormore signal generating moieties, comprise one or more fluorophors,biofluorescent proteins, quantum dots, Raman particles, or combinationsor derivatives thereof; u) the one or more signal generating moietiesprovides an enhanced signal that minimizes detection errors frombackground noise; v) the one or more molecular probes and/or the one ormore detectable components further comprise a spacer group, comprising apolymerized ethylene oxide, a PEG, or a PEO; w) the modified bindingmoiety, the modified scaffold, the oligonucleotide sequence, and/or thecomplementary oligonucleotide sequence comprise HyNic or 4-FB; x) theone or more molecular probes comprise unique, distinguishable, and/orspecifically designed oligonucleotide sequences; y) the one or moredetectable components comprise unique, distinguishable, and/orspecifically designed complementary oligonucleotide sequences; z) theoligonucleotide sequences of the one or more molecular probes areuniquely and specifically designed to hybridize to the complementaryoligonucleotide sequence of the one or more detectable components; a2)the oligonucleotide sequences and/or complementary oligonucleotidesequences, comprise 3′-oligonucleotides, 5′-oligonucleotides, LNAs,PNAs, or combinations or derivatives thereof.

Example 84A

A method for detecting one or more biological targets of a complexsample in a detection assay, comprising: i) preparing and isolating oneor more molecular probes, comprising a binding moiety conjugated to anoligonucleotide sequence, comprising: a) introducing a modified bindingmoiety into a buffered solution; b) conjugating the modified bindingmoieties with at least one modified oligonucleotide at greater than 90%efficiency to form binding moiety-oligonucleotide conjugates; and c)isolating the binding moiety-oligonucleotide conjugates from theconjugation solution by binding the conjugates to an immobilized binder,removing the unconjugated oligonucleotide in a wash step followed byrelease of the bound conjugate from the solid support; and ii) preparingand isolating one or more detectable components, comprising a scaffold,comprising one or more signal generating moieties, conjugated to anoligonucleotide sequence, comprising: a) introducing a modifiedscaffold, comprising the one or more signal generating moieties, into abuffered solution; b) conjugating the modified scaffold with at leastone modified oligonucleotide at greater than 90% efficiency to formscaffold-oligonucleotide conjugates; and c) isolating thescaffold-oligonucleotide conjugates from the conjugation solution bybinding the conjugates to an immobilized binder, removing theunconjugated oligonucleotide in a wash step followed by release of thebound conjugate from the solid support; and iii) providing at least afirst molecular probe and a second molecular probe, comprising a firstbinding moiety conjugated to a first oligonucleotide sequence and asecond binding moiety conjugated to a second oligonucleotide sequence,respectively, to the complex sample comprising the one or morebiological targets; iv) specifically binding the one or more biologicaltargets with the binding moiety of the first molecular probe and thebinding moiety of the second molecular probe; v) providing a universaladapter to the complex sample, wherein the universal adapter comprisedan oligonucleotide sequence having a first sequence segmentcomplementary to the oligonucleotide sequence of the molecular probe anda second sequence segment complementary to the oligonucleotide sequenceof the detectable component; vi) hydridizing the first oligonucleotidesequence of the first target-bound molecular probe to a first portion ofthe first oligonucleotide sequence segment of the universal adapter;vii) hydridizing the second oligonucleotide sequence of the secondtarget-bound molecular probe to a second portion of the firstoligonucleotide sequence segment of the universal adapter; viii)providing the one or more detectable components to the complex sample;ix) hydridizing the oligonucleotide sequences of the one or moredetectable components to the first and second portions of the secondoligonucleotide sequence segment of the universal adapter; and x)detecting one or more signals generated from the hydridized one or moredetectable components; wherein: a) the conjugation between theoligonucleotide sequence and the binding moiety and conjugation betweenthe complementary oligonucleotide sequence and the scaffold, comprises acovalent bond linkage, comprising a hydrazone, oxime, triazine, or otherbond, wherein the formation of the conjugates are at least 90%efficient; b) the binding moiety comprises a binding affinity of lessthan 10⁻⁴ M for the one or more biological targets; c) the hydridizationefficiency of the oligonucleotide sequence to the complementaryoligonucleotide sequence is at least 50%; d) the one or more molecularprobes have substantially the same solubility as the one or more bindingmoieties, respectively; e) the one or more molecular probes comprisemolecular weights of between about 15,000 Daltons to about 450,000Daltons; f) the method of detection generates less false positives thansecondary antibody detection methods; g) the prepared and isolated oneor more molecular probes and one or more detectable components have atleast 90% purity; h) the solubility of the one or more molecular probesminimizes non-specific binding to the one or more biological targets; i)the sample is homogeneous or heterogenous mixture; j) the samplecomprises biological fluids or fluidized biological tissue; k) thesample comprises cells, membranes, biological molecules, metabolites, ordisease biomarkers; l) the sample comprises a range of analytes having awide range of binding specificities; m) the time of conducting themethod, from the start of preparation to the end of detection is betweenabout 2-3 hours; n) the one or more molecular probes are neutral incharge; o) the scaffold comprises a dendrimer, polysaccharide, or adextran; p) the one or more molecular probes comprise a range ofspecificities for the one or more biological targets; q) the detectionassay comprises a singleplex or multiplex detection assay, comprisingimmunodetection, flow cytometry, immunohistochemistry, microspcopy,imaging, high content screening (HCS), ELISA, ELISpot, arrays, beadarrays, or combinations or derivatives thereof; r) the binding moietycomprises an antibody, a protein, a peptide, a carbohydrate, a nuclearreceptor, a small molecule, or combinations or derivatives thereof; s)the one or more biological target comprises an antigen, a pathogen, aprotein, a peptide, an epitope, a carbohydrate-containing molecule, asmall molecule, or combinations or derivatives thereof; t) the one ormore signal generating moieties, comprise one or more fluorophors,biofluorescent proteins, quantum dots, Raman particles, or combinationsor derivatives thereof; u) the one or more signal generating moietiesprovides an enhanced signal that minimizes detection errors frombackground noise; v) the one or more molecular probes and/or the one ormore detectable components further comprise a spacer group, comprising apolymerized ethylene oxide, a PEG, or a PEO; w) the modified bindingmoiety, the modified scaffold, the oligonucleotide sequence, and/or thecomplementary oligonucleotide sequence comprise HyNic or 4-FB; x) theone or more molecular probes comprise unique, distinguishable, and/orspecifically designed oligonucleotide sequences; y) the one or moredetectable components comprise unique, distinguishable, and/orspecifically designed complementary oligonucleotide sequences; z) theoligonucleotide sequences of the one or more molecular probes areuniquely and specifically designed to hybridize to the complementaryoligonucleotide sequence of the one or more detectable components; anda2) the oligonucleotide sequences and/or complementary oligonucleotidesequences, comprise 3′-oligonucleotides, 5′-oligonucleotides, LNAs,PNAs, or combinations or derivatives thereof.

Example 85A

A method for detecting one or more biological targets in a detectionassay, comprising: i) providing at least a first and a second molecularprobe, each comprising a binding moiety conjugated to an oligonucleotidesequence, to a sample comprising the one or more biological targets; ii)binding the one or more biological targets with the binding moiety ofthe first molecular probe and the binding moiety of the second molecularprobe; iii) optionally hydridizing the oligonucleotide sequence of thefirst target-bound molecular probe to a complementary oligonucleotidesequence conjugated to a bead; iv) hydridizing the oligonucleotidesequence of the second target-bound molecular probe to a complementaryoligonucleotide sequence conjugated to a detectable component; and v)detecting a signal generated from the hydridized detectable component;wherein: a) each binding moiety has a binding affinity for the one ormore biological targets; and b) the conjugation between the respectiveoligonucleotide or complementary oligonucleotide sequences and thebinding moiety of the first molecular probe, the binding moiety of thesecond molecular probe, the bead, or the detectable component, comprisesa covalent bond linkage, comprising a hydrazone, oxime, triazine, orother bond, wherein the formation of the conjugate is at least 90%efficient.

Example 86A

A method for detecting one or more biological targets in a detectionassay, comprising: i) providing a first molecular probe, comprising afirst binding moiety conjugated to a first oligonucleotide sequence, toa sample comprising the one or more biological targets; ii) binding theone or more biological targets via the first binding moiety of the firstmolecular probe; iii) providing a second molecular probe, comprising asecond binding moiety conjugated to a second oligonucleotide sequence,to the sample comprising the one or more biological targets; iv) bindingthe one or more biological targets via the second binding moiety of thesecond molecular probe; v) optionally hydridizing the firstoligonucleotide sequence of the first target-bound molecular probe to acomplementary oligonucleotide sequence conjugated to a bead; vi)hydridizing the second oligonucleotide sequence of the secondtarget-bound molecular probe to a complementary oligonucleotide sequenceconjugated to a detectable component; and vii) detecting a signalgenerated from the hydridized detectable component; wherein: a) eachbinding moiety has a binding affinity for the one or more biologicaltargets; b) the conjugation between the respective oligonucleotide orcomplementary oligonucleotide sequences and the first binding moiety,the second binding moiety, the bead, or the detectable component,comprises a covalent bond linkage, comprising a hydrazone, oxime,triazine, or other bond, wherein the formation of the conjugate is atleast 90% efficient.

Example 87A

A method for crosslinking, comprising: i) introducing to a samplecomprising one or more targets: a) one or more firstantibody-oligonucleotide conjugates, comprising a first antibodyconjugated to a first oligonucleotide sequence; and b) one or moresecond antibody-oligonucleotide conjugates, comprising a second antibodyconjugated to a second oligonucleotide sequence; ii) binding the one ormore targets with the first antibody of the one or more firstantibody-oligonucleotide conjugates and with the second antibody of theone or more second antibody-oligonucleotide conjugates to form one ormore sandwich-complexes; iii) contacting the one or moresandwich-complexes with: a) one or more first bead-oligonucleotideconjugate, comprising a first bead conjugated to a complementary firstoligonucleotide sequence; and b) one or more second bead-oligonucleotideconjugate, comprising a second bead conjugated to a complementary secondoligonucleotide sequence; iv) crosslinking the one or moresandwich-complexes by: a) hybridizing the first oligonucleotidesequences of the one or more sandwich-complexes with the complementaryfirst oligonucleotide sequences of the one or more firstbead-oligonucleotide conjugates; and b) hybridizing the secondoligonucleotide sequences of the one or more sandwich-complexes with thecomplementary second oligonucleotide sequences of the one or more secondbead-oligonucleotide conjugates.

Example 88A

The crosslinking method of Example 87A, wherein the formation of thecrosslinked one or more sandwich-complexes forms an agglutination.

Example 89A

The crosslinking method of one or more of Examples 87A-88A, wherein themethod further comprises detecting and/or measuring the degree of theformed agglutination to determine the amount of the one or more targetsin the sample.

Example 90A

The crosslinking method of one or more of Examples 87A-89A, wherein thefirst antibody or the second antibody comprise a monoclonal antibody ora polyclonal antibody.

Example 91A

The crosslinking method of one or more of Examples 87A-90A, wherein thefirst antibody comprises a first monoclonal antibody and the secondantibody comprises a second monoclonal antibody.

Example 92A

The crosslinking method of Example 91A, wherein the first monoclonalantibody is raised against a first epitope of the target and the secondmonoclonal antibody is raised against a second epitope of the target.

Example 93A

The crosslinking method of one or more of Examples 87A-92A, wherein thefirst antibody comprises a first polyclonal antibody and the secondantibody comprises a second polyclonal antibody.

Example 94A

The crosslinking method of Example 93A, wherein a first portion of thefirst polyclonal antibody binds to a first epitope of the target and asecond portion of the second polyclonal antibody binds to a secondepitope of the target.

Example 95A

The crosslinking method of one or more of Examples 89A-94A, wherein thedetection comprises a singleplex or multiplex detection, comprising:immunodetection, flow cytometry, immunohistochemistry, microspcopy,imaging, high content screening (HCS), ELISA, ELISpot, arrays, beadarrays, or combinations or derivatives thereof.

Example 96A

A method of preparing a detectable component having one or moresignal-generating moieties, comprising: i) modifying a scaffold withS-HyNic to form a HyNic-modified scaffold; ii) conjugating a4FB-modified oligonucleotide to the HyNic-modified scaffold, wherein theconjugation is at least 90% efficient; and iii) modifying the scaffoldof the oligonucleotide-scaffold conjugate with one or moresignal-generating moieties to form the detectable component.

Example 97A

A method of preparing one or more components, comprising: i) modifyingone or more scaffolds; ii) conjugating one or more modifiedoligonucleotides to the one or more modified scaffolds, wherein theconjugation is at least 90% efficient; and iii) modifying the scaffoldof the one or more oligonucleotide-scaffold conjugates with one or moresignal-generating moieties to form one or more components; wherein theconjugation between the one or more modified-oligonucleotides and theone or more modified scaffolds comprises a covalent bond linkage,comprising a hydrazone, oxime, triazine, or other bond.

Example 98A

The preparation method of Example 97A, wherein the one or more scaffoldscomprises: a hydrophilic polymer, a dendrimer, a bead, or combinationsthereof.

Example 99A

The preparation method of one or more of Examples 97A-98A, wherein thehydrophilic polymer comprises a polysaccharide molecule.

Example 100A

The preparation method of Example 99A wherein the polysaccharidemolecule comprises a dextran or an amino-dextran.

Example 101A

The preparation method of one or more of Examples 97A-100A, wherein theone or more signal-generating moieties comprises a directly detectablesignal-generating moiety or an indirectly detectable signal-generatingmoiety.

Example 102A

The preparation method of Example 101A, wherein the directly detectablesignal-generating moiety comprises: a fluorescent dye; a luminescentspecies; a phosphorescent species; a radioactive substance; ananoparticle; a diffracting particle; a raman particle; a metalparticle; a magnetic particle; a bead; an RFID tag; a microbarcodeparticle; or combinations thereof.

Example 103A

The preparation method of one or more of Examples 101A-102A, wherein theindirectly detectable signal-generating moiety comprises: an enzyme; anantibody; an antigen; a nucleic acid; a nucleic acid analog; a ligand; asubstrate; a hapten; or combinations thereof.

Example 104A

The preparation method of one or more of Examples 97A-103A, wherein theone or more scaffolds signal-generating moieties comprise: afluorophore; a chromophore; a biofluorescent protein; a fluorophorelabeled DNA dendrimer; a Quantum Dot; a chemiluminescent compound; aelectrochemiluminescent label; a bioluminescent label; a polymer; apolymer particle; a bead; a Raman particle; a heavy metal chelate; goldor other metal particles or heavy atoms; a spin label; a radioactiveisotope; a secondary reporter; a hapten; a nucleic acid or nucleic acidanalog; a protein; a peptide ligand or substrate; a receptor; an enzyme;an enzyme substrate; an antibody; an antibody fragment; an antigen; orcombinations or derivatives thereof. In addition, the preparationmethods of one or more of Examples 97A-103A, wherein the one or morescaffolds signal-generating moieties may also comprise: polymer-basedheavy metal chelates conjugated to antibodies and other binders may alsobe used to multiplex protein analysis using a technique named CyTOF(CYtometry Time Of Flight). Heavy metal isotopes of Ru, Rh, Pd, Ag, In,La, Hf, Re, Ir, Pt, Au, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yband Lu can be used.

Example 105A

A method for binding, comprising: i) providing at least a firstmolecular probe and a second molecular probe to a sample comprising aplurality of cells, wherein the first binding moiety is conjugated to afirst oligonucleotide sequence and a second binding moiety is conjugatedto a second oligonucleotide sequence; ii) specifically binding a firsttarget of a first cell and a second target of a second cell of theplurality of cells, comprising: a) binding the first target with thefirst binding moiety of the first molecular probe; and b) binding thesecond target with the second binding moiety of the second molecularprobe; iii) providing to the sample one or more detectable components,comprising: a) a first detectable component, comprising one or moresignal generating moieties conjugated to a first oligonucleotidecomplementary to the first oligonucleotide sequence of the firstmolecular probe; and b) a second detectable component, comprising one ormore signal generating moieties conjugated to a second oligonucleotidecomplementary to the second oligonucleotide sequence of the secondmolecular probe; iv) hybridizing the first oligonucleotide sequence ofthe first bound molecular probe to the complementary firstoligonucleotide sequence of the first detectable component; v)hybridizing the second oligonucleotide sequence of the second boundmolecular probe to the complementary second oligonucleotide sequence ofthe second detectable component; and vi) detecting by flow cytometry theone or more signals generated from the first hybridized detectablecomponent and the second hybridized detectable component; wherein:

a) the conjugation of the first molecular probe and the second molecularprobe and the conjugation of the first detectable component and thesecond detectable component comprise a covalent bond linkage, comprisinga hydrazone, oxime, triazine, or other bond; b) the formation of theconjugates are at least 90% efficient; and c) the first binding moietyhas a binding affinity for the first target of less than 10⁻⁴ M and thesecond binding moiety has a binding affinity for the second target ofless than 10⁻⁴ M.

Example 106A

The binding method of Example 105A, wherein the first target comprises afirst biomarker of the first cell and the second target comprises asecond biomarker of the second cell.

Example 107A

The binding method of one or more of Examples 105A-106A, wherein thefirst biomarker comprises a protein biomarker and the second biomarkercomprises a protein biomarker.

Example 108A

The binding method of one or more of Examples 106A-107A, wherein thefirst biomarker comprises an adhesion molecule and the second biomarkercomprises an adhesion molecule.

Example 109A

The binding method of one or more of Examples 105A-108A, wherein theplurality of cells comprises spleenocytes.

Example 110A

The binding method of one or more of Examples 105-109, wherein the firstcell is a T-cell and the second cell is a B-cell.

Example 111A

A method for binding, comprising: i) electrophoresing material derivedfrom lysing a plurality of cells; ii) transferring the electrophoresedmaterial to a membrane; iii) incubating the membrane with at least onemolecular probe, comprising a binding moiety conjugated to anoligonucleotide sequence; iv) specifically binding at least one targetof the electrophoresed material with the binding moiety of the at leastone molecular probe; v) further incubating the membrane with at leastone detectable component, comprising one or more signal generatingmoieties conjugated to an oligonucleotide complementary to theoligonucleotide sequence of the at least one molecular probe; and vi)hybridizing the oligonucleotide sequence of the at least one boundmolecular probe to the complementary oligonucleotide sequence of the atleast one detectable component; wherein: a) the conjugation of the atleast one molecular probe and the conjugation of the at least onedetectable component comprise a covalent bond linkage, comprising ahydrazone, oxime, triazine, or other bond; b) the formation of theconjugates are at least 90% efficient; and c) the binding moiety of theat least one molecular probe has a binding affinity for the at least onetarget of less than 10⁻⁴ M.

Example 112A

The binding method of Example 111, wherein the membrane comprises a PVDFmembrane.

Example 113A

The binding method of one or more of Examples 111A-112A, wherein the oneor more signal generating moieties of the at least one detectablecomponent comprises horseradish peroxidase.

Example 114A

The binding method of one or more of Examples 111A-113A, wherein theelectrophoresing of the lysate comprises a Western Blot.

Example 115A

The binding method of one or more of Examples 111A-114A, wherein themethod comprises detecting the at least one signal generated from theone or more signal generating moieties on the at least one hybridizeddetectable component.

Example 116A

The binding method of one or more of Examples 111A-115A, wherein thedetection comprises a singleplex or multiplex detection, comprising:immunodetection, flow cytometry, chemiluminescence detection, infrareddetection, immunohistochemistry, ELISA, ELISpot, arrays, bead arrays, orcombinations or derivatives thereof.

Example 117A

A method of binding one or more targets, comprising: i) preparing aplurality molecular probes, comprising at least: a) conjugating auniversal oligonucleotide sequence to a first binding moiety to form afirst molecular probe; and b) conjugating a universal oligonucleotidesequence to a second binding moiety to form a second molecular probe;ii) hybridizing the plurality of formed molecular probes with aplurality of universal adapters, comprising: a) introducing a firstuniversal adapter to the first molecular probe, wherein the firstuniversal adapter comprises an oligonucleotide sequence comprising: i) acomplementary universal sequence segment; and ii) a first sequencesegment; b) hybridizing the universal oligonucleotide sequence of thefirst molecular probe to the complementary universal sequence segment ofthe first universal adapter; c) introducing a second universal adapterto the second molecular probe, wherein the second universal adaptercomprises an oligonucleotide sequence comprising: i) a complementaryuniversal sequence segment; and ii) a second sequence segment; d)hybridizing the universal oligonucleotide sequence of the secondmolecular probe to the complementary universal sequence segment of thesecond universal adapter; and iii) introducing the plurality ofhybridized molecular probes to a sample comprising one or more targets;iv) binding the one or more targets with the plurality of hybridizedmolecular probes, comprising: a) binding a first target of the one ormore targets with the first binding moiety of the first hybridizedmolecular probe; b) binding a second target of the one or more targetswith the second binding moiety of the second hybridized molecular probe;v) introducing to the sample a plurality of detectable components,comprising: a) a first detectable component comprising one or moresignal generating moieties conjugated to a first oligonucleotidesequence complementary to the first sequence segment of the firstuniversal adapter; and b) a second detectable component comprising oneor more signal generating moieties conjugated to a secondoligonucleotide sequence complementary to the second sequence segment ofthe second universal adapter; and vi) hybridizing the plurality of boundmolecular probes with the plurality of detectable components,comprising: a) hybridizing the first sequence segment of the first boundmolecular probe to the complementary first oligonucleotide sequence ofthe first detectable component; and b) hybridizing the second sequencesegment of the second bound molecular probe to the complementary secondoligonucleotide sequence of the second detectable component; wherein: a)the conjugation of the plurality of molecular probes and the conjugationof the plurality of the detectable components comprise a covalent bondlinkage, comprising a hydrazone, oxime, triazine, or other bond; b) theformation of the conjugates are at least 90% efficient; and c) the firstbinding moiety has a binding affinity for the first target of less than10⁻⁴ M and the second binding moiety has a binding affinity for thesecond target of less than 10⁻⁴ M.

Example 118A

The binding method of Example 117A, wherein the method further comprisesdetecting a signal generated from the hybridized detectable component.

Example 119A

The binding method of one or more of Examples 117A-118A, wherein thedetectable component comprises a scaffold comprising one or more signalgenerating moieties.

Example 120A

The binding method of Example 119A, wherein the scaffold is conjugatedto the oligonucleotide sequence.

Example 121A

The binding method of one or more of Examples 117A-120A, wherein themethod comprises a singleplex or multiplex detection assay, comprisingimmunodetection, flow cytometry, immunohistochemistry, microspcopy,imaging, high content screening (HCS), ELISA, ELISpot, arrays, beadarrays, or combinations or derivatives thereof.

Example 122A

The binding method of Example 121A, wherein the target comprises cellsand/or cellular components.

Example 123A

The binding method of Example 122A, wherein the cells are attached to abead or a plate.

Example 124A

A method for binding one or more targets, comprising: i) forming atleast one molecular probe by conjugating a universal oligonucleotidesequence to one or more binding moieties; ii) hybridizing the formed atleast one molecular probe with at least one universal adapter,comprising: a) introducing the at least one universal adapter to theformed at least one molecular probe, wherein the at least one universaladapter comprises an oligonucleotide sequence comprising: i) acomplementary universal sequence segment; and ii) a first sequencesegment; and b) hybridizing the universal oligonucleotide sequence ofthe formed at least one molecular probe to the complementary universalsequence segment of the at least one universal adapter; iii) introducingthe hybridized at least one molecular probe to a sample comprising theone or more targets; iv) binding at least one of the one or more targetswith the binding moiety of the hybridized at least one molecular probe;v) introducing to the sample at least one detectable componentcomprising one or more signal generating moieties conjugated to anoligonucleotide sequence complementary to the first sequence segment ofthe at least one universal adapter; and vi) hybridizing the firstsequence segment of the bound at least one molecular probe to thecomplementary oligonucleotide sequence of the at least one detectablecomponent; wherein: a) the conjugation of the at least one molecularprobe and the conjugation of the at least one detectable componentcomprise a covalent bond linkage, comprising a hydrazone, oxime,triazine, or other bond; b) the formation of the conjugates are at least90% efficient; and c) the binding moiety has a binding affinity for thetarget of less than 10⁻⁴ M.

Example 125A

The binding method of Example 124A, wherein the one or more targetscomprises cells, cellular components, biomarkers, biological components,or combinations thereof.

Example 126A

The binding method of Example 125A, wherein the cells are attached to abead or a plate.

Example 127A

The binding method of one or more of Examples 125A-126A, wherein thecellular components comprises tubulin.

Example 128A

The binding method of one or more of Examples 124A-127A, wherein the atleast one detectable component comprises a scaffold comprising one ormore signal generating moieties.

Example 129A

The binding method of one or more of Examples 124A-128A, wherein thehybridized at least one detectable component generates a signal.

Example 130A

The binding method of Example 129A, wherein signal generated by thehybridized at least one detectable component is detected.

Example 131A

The binding method of Example 130A, wherein the method comprises asingleplex or multiplex detection assay, comprising immunodetection,flow cytometry, immunohistochemistry, microspcopy, imaging, high contentscreening (HCS), ELISA, ELISpot, arrays, bead arrays, or combinations orderivatives thereof.

Example 132A

A method for detecting a target, comprising: i) conjugating a bindingmoiety to an oligonucleotide sequence to form a molecular probe; ii)introducing the molecular probe to a universal adapter, wherein theuniversal adapter comprises an oligonucleotide sequence having: a) afirst sequence segment complementary to the oligonucleotide sequence ofthe molecular probe; and b) a second sequence segment; iii) hybridizingthe oligonucleotide sequence of the molecular probe to the complementaryfirst sequence segment of the universal adapter; iv) introducing thehybridized molecular probe to a sample comprising the target; v) bindingthe target with the binding moiety of the hybridized molecular probe;vi) introducing to the sample comprising the bound molecular probe adetectable component, wherein the detectable component comprises acomplementary oligonucleotide sequence to the second sequence segmentconjugated to one or more signal generating moieties; vii) hybridizingthe second sequence segment of the molecular probe to the complementaryoligonucleotide sequence of the detectable component; and viii)detecting signal generated from the hybridized detectable component;wherein: a) the conjugation of the molecular probe and the detectablecomponent independently comprise a covalent bond linkage, comprising ahydrazone, oxime, triazine, or other bond; b) the formation of theconjugates are at least 90% efficient; and c) the binding moiety has abinding affinity for the biological target of less than 10⁻⁴ M.

Example 133A

A method for detecting one or more targets, comprising: i) providing atleast a first molecular probe and a second molecular probe to a samplecomprising the one or more targets,

wherein the first binding moiety is conjugated to a firstoligonucleotide sequence and a second binding moiety is conjugated to asecond oligonucleotide sequence; ii) specifically binding a first targetand a second target of the one or more targets, comprising: a) bindingthe first target with the first binding moiety of the first molecularprobe; and b) binding the second target with the second binding moietyof the second molecular probe; iii) providing to the sample one or moredetectable components, comprising: a) a first detectable component,comprising a first bead, having one or more signal generating moieties,conjugated to a first oligonucleotide complementary to the firstoligonucleotide sequence of the first molecular probe; and b) a seconddetectable component, comprising a second bead, having one or moresignal generating moieties conjugated to a second oligonucleotidecomplementary to the second oligonucleotide sequence of the secondmolecular probe; iv) hybridizing the first oligonucleotide sequence ofthe first bound molecular probe to the complementary firstoligonucleotide sequence of the first detectable component; v)hybridizing the second oligonucleotide sequence of the second boundmolecular probe to the complementary second oligonucleotide sequence ofthe second detectable component; and vi) detecting the one or moresignals generated from at least the first hybridized detectablecomponent and the second hybridized detectable component; wherein: a)the conjugation of the plurality of molecular probes and the conjugationof the plurality of the detectable components comprise a covalent bondlinkage, comprising a hydrazone, oxime, triazine, or other bond; b) theformation of the conjugates are at least 90% efficient; and c) the firstbinding moiety has a binding affinity for the first target of less than10⁻⁴ M and the second binding moiety has a binding affinity for thesecond target of less than 10⁻⁴ M.

Example 134A

A method for detecting one or more targets, comprising: i) providing atleast a first molecular probe and a second molecular probe to a samplecomprising the one or more targets, wherein the first binding moiety isconjugated to a first oligonucleotide sequence and a second bindingmoiety is conjugated to a second oligonucleotide sequence; ii)specifically binding a first target and a second target of the one ormore targets, comprising: a) binding the first target with the firstbinding moiety of the first molecular probe; and b) binding the secondtarget with the second binding moiety of the second molecular probe;iii) providing to the sample one or more detectable components,comprising: a) a first detectable component, comprising a first bead,having one or more signal generating moieties, conjugated to a firstoligonucleotide complementary to the first oligonucleotide sequence ofthe first molecular probe; and b) a second detectable component,comprising one or more signal generating moieties conjugated to a secondoligonucleotide complementary to the second oligonucleotide sequence ofthe second molecular probe; iv) hybridizing the first oligonucleotidesequence of the first bound molecular probe to the complementary firstoligonucleotide sequence of the first detectable component; v)hybridizing the second oligonucleotide sequence of the second boundmolecular probe to the complementary second oligonucleotide sequence ofthe second detectable component; and vi) detecting the one or moresignals generated from at least the first hybridized detectablecomponent and the second hybridized detectable component; wherein: a)the conjugation of the plurality of molecular probes and the conjugationof the plurality of the detectable components comprise a covalent bondlinkage, comprising a hydrazone, oxime, triazine, or other bond; b) theformation of the conjugates are at least 90% efficient; and c) the firstbinding moiety has a binding affinity for the first target of less than10⁻⁴ M and the second binding moiety has a binding affinity for thesecond target of less than 10⁻⁴ M.

Example 135A

The binding method of Example 134A, wherein the second detectablecomponent comprises a scaffold comprising the one or more signalgenerating moieties.

Example 136A

The binding method of Example 135A, wherein the scaffold is conjugatedto the complementary second oligonucleotide sequence.

Example 137A

The method of one or more of Examples 87A-136A, wherein theoligonucleotide sequences and/or complementary oligonucleotidesequences, comprise 3′-oligonucleotides, 5′-oligonucleotides, LNAs,PNAs, or combinations or derivatives thereof.

Example 1B

A purification method, comprising: i) providing a sample comprising atleast one of the following: a) a carbonyl modified-molecule; and b) acarbonyl modified-biomolecule; ii) contacting the sample with a solidsupport comprising an acyl hydrazide group; iii) binding the carbonylmodified-molecule or the carbonyl modified-biomolecule to the acylhydrazide group of the solid support; and iv) recovering a purifiedcarbonyl modified-molecule or carbonyl modified-biomolecule bycontacting the bound-carbonyl modified-molecule or the bound-carbonylmodified-biomolecule with a mixture comprising an aromatic or aliphaticcarbonyl molecule and an aniline-containing compound.

Example 2B

The purification method of Example 1B, wherein the binding stepcomprises chemoselectively binding the carbonyl modified-molecule or thecarbonyl modified-biomolecule to the acyl hydrazide group of the solidsupport.

Example 3B

The purification method of Examples 1B or 2B, wherein the method furthercomprises removing unmodified-molecule or unmodified-biomolecule fromthe bound-carbonyl modified-molecule or the bound-carbonylmodified-biomolecule by washing the solid support.

Example 4B

The purification method of any one of Examples 1B-3B, wherein therecovering step comprises releasing the chemoselectively bound-carbonylmodified-molecule or the chemoselectively bound-carbonylmodified-biomolecule from the solid support.

Example 5B

The purification method of any one of Examples 1B-4B, wherein thecontacting with a mixture comprising an aromatic or aliphatic carbonylmolecule and an aniline-containing compound comprises incubating.

Example 6B

The purification method of any one of Examples 1B-5B, wherein thecarbonyl modified-molecule is an aromatic carbonyl modified-molecule.

Example 7B

The purification method of any one of Examples 1B-6B, wherein thecarbonyl modified-biomolecule is an aromatic carbonylmodified-biomolecule.

Example 8B

The purification method of any one of Examples 1B-7B, wherein thecarbonyl moiety is an aromatic aldehyde.

Example 9B

The purification method of any one of Examples 1B-8B, wherein thecarbonyl modified-molecule or the carbonyl modified-biomolecule is a4FB-oligonucleotide.

Example 10B

The purification method of any one of Examples 1B-9B, wherein thearomatic aldehyde is 4-formylbenzamide

Example 11B

The purification method of any one of Examples 1B-10B, wherein the acylhydrazide group is immobilized or bound to the solid support.

Example 12B

The purification method of any one of Examples 1B-11B, wherein theimmobilized hydrazide moiety of the acyl hydrazide group is an aliphatichydrazide.

Example 13B

The purification method of any one of Examples 1B-12B, wherein theimmobilized hydrazide moiety of the acyl hydrazide group is an aromatichydrazide.

Example 14B

The purification method of any one of Examples 1B-13B, wherein thereleasing aromatic carbonyl molecule is 2-sulfo-benzaldehyde.

Example 15B

The purification method of any one of Examples 1B-14B, wherein themixture utilized to conduct the releasing step comprises an aqueousbuffer.

Example 16B

The purification method of any one of Examples 1B-15B, wherein themixture utilized to conduct the releasing step comprises an aqueousbuffer having an approximate pH 2.0-8.0.

Example 17B

The purification method of any one of Examples 1B-16B, wherein themethod further comprises exchanging the release buffer for a suitablebuffer or water.

Example 18B

The purification method of any one of Examples 1B-17B, wherein themixture utilized to conduct the releasing step comprises the aromaticcarbonyl molecule in a solution of 1-300 mM aniline.

Example 19B

The purification method of any one of Examples 1B-18B, wherein themixture utilized to conduct the releasing step comprises the aromaticcarbonyl molecule in a solution of 1-300 mM aniline in an aqueousbuffer.

Example 20B

The purification method of any one of Examples 1B-19B, wherein themixture utilized to conduct the releasing step comprises the aromaticcarbonyl molecule in a solution of 1-300 mM aniline in an aqueous bufferhaving an approximate pH 2.0-8.0.

Example 21B

The purification method of any one of Examples 1B-20B, wherein therelease buffer comprises 100 mM phosphate.

Example 22B

The purification method of any one of Examples 1B-21B, wherein therelease buffer comprises 150 mM NaCl.

Example 23B

The purification method of any one of Examples 1B-22B, wherein therelease buffer comprises a pH of about 5.0.

Example 24B

The purification method of any one of Examples 1B-23B, wherein therelease buffer comprises 25 mM aniline.

Example 25B

The purification method of any one of Examples 1B-24B, wherein thecarbonyl modified-molecule comprises a detectable component.

Example 26B

The purification method of any one of Examples 1B-25B, wherein thecarbonyl modified-molecule comprises a detectable component comprisingan oligonucleotide conjugated to one or more signal generating moieties.

Example 27B

The purification method of any one of Examples 1B-26B, wherein thecarbonyl modified-molecule comprises a detectable component comprisingan oligonucleotide conjugated to at least one scaffold comprising one ormore signal generating moieties.

Example 28B

The purification method of any one of Examples 1B-27B, wherein thescaffold comprises a dendrimer, a polysaccharide, a dextran, a protein,a peptide, a further oligonucleotide sequence, a polymer, a hydrophilicpolymer, a bead, a nanoparticle, or combinations or derivatives thereof.

Example 29B

The purification method of any one of Examples 1B-28B, wherein one ormore signal generating moieties comprises one or more of the following:a directly detectable signal generating moiety, an indirectly detectablesignal generating moiety, a fluorescent dye, a fluorophore, afluorochrome, a chromophore, a biofluorescent protein, a luminescentspecies, a chemiluminescent compound, a electrochemiluminescent label, abioluminescent label, a phosphorescent species, a fluorophore labeledDNA dendrimer, Quantum Dot, a tandem dye, a FRET dye, a heavy atom, aspin label, a radioactive isotope, a nanoparticle, a light scatteringnanoparticle or microsphere, a diffracting particle, a polymer, apolymer particle, a bead, a solid surface, a Raman particle, a metalparticle, a stable isotope, a heavy metal chelate, a magnetic particle,a bead, an RFID tag, a microbarcode particle, an enzyme, an enzymesubstrate, a molecule specifically recognized by another substancecarrying a label or reacts with a substance carrying a label, anantibody, an antibody fragment, an antigen, a nucleic acid, a nucleicacid analog, oligonucleotide, oligonucleotide analog, complementaryoligonucleotide, complementary oligonucleotide analog, a ligand, aprotein, a peptide ligand, a protein substrate, a receptor; a substrate,a secondary reporter, a hapten, or combinations thereof.

Example 30B

The purification method of any one of Examples 1B-29B, wherein thecarbonyl modified-biomolecule comprises molecular probe.

Example 31B

The purification method of any one of Examples 1B-30B, wherein thecarbonyl modified-biomolecule comprises molecular probe comprising abinding moiety conjugated to at least one oligonucleotide.

Example 32B

The purification method of any one of Examples 1B-31B, wherein thebinding moiety comprises an antibody, a monoclonal antibody, apolyclonal antibody, an enzyme, a protein, a peptide, a carbohydrate, anuclear receptor, a small molecule, an aptamer, a chelator, orcombinations or derivatives thereof.

Example 33B

The purification method of any one of Examples 1B-32B, wherein themolecular probe and/or the detectable component further comprises aspacer group, comprising a polymerized ethylene oxide, a PEG, a PEO, aprotein, a peptide, a DNA, an RNA, an oligonucleotide sequence, or adextran.

Example 34B

The purification method of any one of Examples 1B-33B, wherein thepurification method purifies a genetically engineered protein.

Example 35B

The purification method of any one of Examples 1B-34B, wherein thegenetically engineered protein comprises an incorporated an aliphatic oraromatic carbonyl group.

Example 36B

The purification method of any one of Examples 1B-35B, wherein thegenetically engineered protein has been produced to incorporate analiphatic or aromatic carbonyl group.

Example 1C

An assay method, comprising: i) providing to a sample comprising aplurality of targets: a) at least a first molecular probe comprising afirst oligonucleotide sequence conjugated to a first binding moietyhaving an affinity for at least a first target of the plurality oftargets; and b) at least a first particle, bead, or other surface,comprising a complementary second oligonucleotide sequence conjugated tosaid at least particle, bead, or other surface; wherein the amount ofthe second complementary oligonucleotide sequence is greater that theamount of the first oligonucleotide sequence; and iii) mixing ormaintaining contact between said at least first molecular probe and saidat least first particle, bead, or other surface, to hybridize all orsubstantially all of the first oligonucleotide sequence of the at leastfirst molecular probe with the second complementary oligonucleotidesequence conjugated to said at least first particle, bead, or othersurface.

Example 2C

The method of Example 1C, wherein the mode of addition comprises: i) theat least first molecular probe and the at least first particle, bead, orother surface, are combined together and hybridized prior to contactingthe sample; ii) the at least first molecular probe is combined with thesample prior to the addition of the at least first particle, bead, orother surface; or iii) the at least first particle, bead, or othersurface, is combined with the sample prior to the addition of the atleast first molecular probe.

Example 3C

The method of Examples 1C or 2C, wherein the method comprises: i) the atleast first molecular probe binding the target prior to hybridizing withthe at least first particle, bead, or other surface; or ii) the at leastfirst molecular probe hybridizing with the at least first particle,bead, or other surface, prior to binding the target.

Example 4C

An assay method, comprising: i) providing to a sample comprising aplurality of targets: a) at least a first molecular probe comprising afirst oligonucleotide sequence conjugated to a first binding moietyhaving an affinity for at least a first target of the plurality oftargets; b) at least a first particle, bead, or other surface,comprising a second oligonucleotide sequence conjugated to said at leastparticle, bead, or other surface; wherein the amount of the secondoligonucleotide sequence is greater that the amount of the firstoligonucleotide sequence; and c) at least a first universal adapter,comprising an oligonucleotide sequence having a first sequence segmentcomplementary to the first oligonucleotide sequence of the at leastfirst molecular probe and a second sequence segment complementary to thesecond oligonucleotide sequence of the at least first particle, bead, orother surface; and iii) mixing or maintaining contact between said atleast first molecular probe, said at least first particle, bead, orother surface, and said at least first universal adapter, to hybridizeall or substantially all of: a) the first oligonucleotide sequence ofthe at least first molecular probe with the first complementaryoligognucleotide sequence segment of the at least first universaladapter; and b) the second oligognucleotide sequence of the at leastfirst particle, bead, or other surface, with the second complementaryoligognucleotide sequence segment of the at least first universaladapter.

Example 5C

The method of Example 4C, wherein the mode of addition comprises: i) theat least first molecular probe, the at least first universal adapter,and the at least first particle, bead, or other surface, are combinedtogether and hybridized prior to contacting the sample; ii) the at leastfirst molecular probe and the at least first universal adapter arecombined together and hybridized prior to contacting the sample; iii)the at least first particle, bead, or other surface, and the at leastfirst universal adapter are combined together and hybridized prior tocontacting the sample; iv) the at least first molecular probe, alone orin combination with the at least first particle, bead, or other surface,is combined with the sample prior to the addition of the at least firstuniversal adapter; or v) the at least first universal adapter iscombined with the sample prior to the addition of the at least firstmolecular probe and/or the at least first particle, bead, or othersurface.

Example 6C

The method of Examples 4C or 5C, wherein the method comprises: i) the atleast first molecular probe hybridizing with the at least firstuniversal adapter prior to said at least first molecular probe bindingthe target; ii) the at least first molecular probe hybridizing with theat least first universal adapter after said at least first molecularprobe binds the target; iii) the at least first particle, bead, or othersurface, hybridizing with the at least first universal adapter prior tothe at least first molecular probe binding the target; iv) the at leastfirst particle, bead, or other surface, hybridizing with the at leastfirst universal adapter after the at least first molecular probe bindsthe target; v) the at least first universal adapter hybridizing with theat least first molecular probe and hybridizing with the at least firstparticle, bead, or other surface, prior to said at least first molecularprobe binding the target; or vi) the at least first universal adapterhybridizing with the at least first molecular probe and hybridizing withthe at least first particle, bead, or other surface, after said at leastfirst molecular probe binds the target.

Example 7C

The method of any one of Examples 1C-6C, wherein the sample furthercomprises a plurality of non-target materials comprising at least afirst non-target material.

Example 8C

The method of any one of Examples 1C-7C, wherein the at least firstnon-target material comprises one or more of the following: non-targetantigens, non-target cells, or non-target cellular components.

Example 9C

The method of any one of Examples 1C-8C, wherein the at least firstmolecular probe is added in an excess amount as compared to the amountof the at least first target present in the sample.

Example 10C

The method of any one of Examples 1C-9C, wherein the at least firstmolecular probe recognizes the at least first target of the plurality oftargets in the presence of the at least first non-target material.

Example 11C

The method of any one of Examples 1C-10C, wherein the at least firstrecognized-target is an antigen, an antigen on a cell surface, acellular component, or a cell; wherein in said recognition is in thepresence of one or more non-target materials.

Example 12C

The method of any one of Examples 1C-11C, wherein the at least firstmolecular probe binds the at least first target of the plurality oftargets in the presence of the at least first non-target material.

Example 13C

The method of any one of Examples 1C-12C, wherein the at least firstbound-target is an antigen, an antigen on a cell surface, a cellularcomponent, or a cell; wherein in said binding is in the presence of oneor more non-target materials.

Example 14C

The method of any one of Examples 1C-13C, wherein the at least firsttarget is a biological target.

Example 15C

The method of any one of Examples 1C-14C, wherein the at least firsttarget is a biological target comprising an antigen, a pathogen, aprotein, a peptide, an epitope, a carbohydrate-containing molecule, asmall molecule, or combinations or derivatives thereof.

Example 16C

The method of any one of Examples 1C-15C, wherein the at least firsttarget is an antigen.

Example 17C

The method of any one of Examples 1C-16C, wherein the first bindingmoiety comprises an antibody, a monoclonal antibody, a polyclonalantibody, an enzyme, a protein, a peptide, a nuclear receptor, or anaptamer.

Example 18C

The method of any one of Examples 1C-17C, wherein the method is a celldepletion method.

Example 19C

The method of any one of Examples 1C-18C, wherein the method is animmunoprecipitation method.

Example 20C

The method of any one of Examples 1C-19C, wherein the complementarysecond oligonucleotide sequence is tethered to the at least firstparticle, bead, or other surface.

Example 21C

The method of any one of Examples 1C-20C, wherein said at least firstparticle, bead, or other surface, comprises at least one of thefollowing: i) a magnetic bead, a paramagnetic bead, or asuperparamagnetic bead; ii) a dense object, a buoyant object, or astationary object; iii) a gel particle or a matrix, said gel particle ormatrix comprising an agarose bead or a sepharose bead; and iv) a filteror a mesh.

Example 22C

The method of any one of Examples 1C-21C, wherein said at least firstparticle, bead, or other surface enables at least one of the following:i) magnetic separation; ii) mechanical separation; iii) chromatographicseparation; and iv) filtration separation.

Example 23C

The method of any one of Examples 1C-22C, wherein the magnetic bead, aparamagnetic bead, or a superparamagnetic bead enables magneticseparation.

Example 24C

The method of any one of Examples 1C-23C, wherein the dense object, abuoyant object, or a stationary object enables mechanical separation.

Example 25C

The method of any one of Examples 1C-24C, wherein the gel particle orthe matrix enables chromatographic separation.

Example 26C

The method of any one of Examples 1C-25C, wherein the filter or meshenables filtration separation.

Example 27C

The method of any one of Examples 1C-26C, wherein the at least firstbound-target is captured by separating said at least first particle,bead, or other surface from the mixture containing the non-targetmaterials.

Example 28C

The method of any one of Examples 1C-27C, wherein the separation removesor excludes the mixture containing the non-target materials.

Example 29C

The method of any one of Examples 1C-28C, wherein the separation removesor excludes the at least first bound-target from the mixture containingthe non-target materials.

Example 30C

The method of any one of Examples 1C-29C, wherein the separated at leastfirst bound-target is further washed to remove residual non-targetmaterials.

Example 31C

The method of any one of Examples 1C-30C, wherein the non-targetmaterials are further washed to collect residual at least firstbound-target.

Example 32C

The method of any one of Examples 1C-31C, wherein the method comprises aseries of sequential or parallel steps to capture or exclude one or moretargets in a sample comprising the plurality of targets.

Example 33C

The method of any one of Examples 1C-32C, wherein: i) the assaycomprises a singleplex or multiplex assay; and ii) the assay furthercomprises detecting, measuring, or quantifying the level of bindingand/or amount of the target present in the sample with one or more ofthe following: flow cytometry, immunomagnetic cellular depletion,immunomagnetic cell capture, array, bead array, multiplex bead array,microarray, antibody array, cellular array, chemiluminescence, infrared,microscopy, imaging, high content screening (HCS), mass cytometry,lateral flow immunoassay, immunodetection, immunohistochemistry (IHC),immunocytochemistry (ICC), in situ hybridization (ISH), enzymeimmuno-assay (EIA), enzyme linked immuno-assay (ELISA), ELISpot,immunoturbidity, latex agglutination, gold particle agglutination,visual inspection, a change in light transmittance through said sample,increased light transmittance through said sample, a blotting method, aWestern blot, a Southern blot, a Southwestern blot, labeling inside anelectrophoresis system, labeling on a surface, labeling on an array, PCRamplification, elongation followed by PCR amplification,immunoprecipitation, co-immunoprecipitation, chromatinimmunoprecipitation, pretargeting imaging, therapeutic agent, orcombinations thereof.

Example 34C

The method of any one of Examples 1C-33C, wherein: i) the assaycomprises a singleplex or multiplex assay; and ii) the assay furthercomprises detecting, measuring, or quantifying the level of bindingand/or amount of the target present in the sample with one or more ofthe following: flow cytometry, microscopy, imaging, high contentscreening (HCS), multiplex bead array, microarray, antibody array,cellular array, immunohistochemistry (IHC), immunocytochemistry (ICC),in situ hybridization (ISH), enzyme immuno-assay (EIA), enzyme linkedimmuno-assay (ELISA), ELISpot, or a blotting method.

Example 35C

The method of any one of Examples 1C-34C, wherein: i) the assaycomprises a singleplex or multiplex assay; and ii) the assay furthercomprises detecting, measuring, or quantifying the level of bindingand/or amount of the target present in the sample with one or more ofthe following: flow cytometry, mass cytometry, lateral flow immunoassay,immunohistochemistry (IHC), immunocytochemistry (ICC),immunoprecipitation, pretargeting imaging, therapeutic agent, orcombinations thereof.

Example 36C

The method of any one of Examples 1C-35C, wherein the sample ischaracterized as at least one or more of the following: i) a complexsample; and ii) a homogeneous or a heterogeneous mixture; wherein saidsample comprises at least one or more of the following: a) one or moreanalytes having substantially the same or substantially differentbinding specificities; b) one or more of the following biologiccomponents, comprising: cells, membranes, biological molecules,metabolites, or disease biomarkers; and c) a biological fluid or afluidized biological tissue.

Example 37C

The method of any one of Examples 1C-36C, wherein the method comprisesone or more of the following: i) the hybridization efficiency of thefirst oligonucleotide sequence to the second oligonucleotide sequence isat least 50% with respect to the at least first particle, bead, or othersurface, under the hybridization conditions employed; ii) thehybridization efficiency of the first oligonucleotide sequence to thecomplementary first oligonucleotide sequence segment is at least 50%with respect to the at least first molecular probe, under thehybridization conditions employed; or iii) the hybridization efficiencyof the second oligonucleotide sequence to the complementary secondoligonucleotide sequence segment is at least 50% with respect to the atleast first particle, bead, or other surface, under the hybridizationconditions employed.

Example 38C

The method of any one of Examples 1C-37C, wherein the at least firstmolecular probe, the at least first particle, bead, or other surface,and/or at least first universal adapter further comprises a spacergroup, comprising a polymerized ethylene oxide, a PEG, a PEO, a protein,a peptide, a DNA, an RNA, an oligonucleotide sequence, or a dextran.

Example 39C

The method of any one of Examples 1C-38C, wherein the conjugation of thefirst binding moiety to the first oligonucleotide sequence comprises aHyNic or a 4-FB residue; and wherein the conjugation of the at leastfirst particle, bead, or other surface, to the second oligonucleotidesequence comprises a HyNic or a 4-FB residue.

Example 40C

The method of any one of Examples 1C-39C, wherein the firstoligonucleotide sequence, second oligonucleotide sequence, and/oroligonucleotide sequence segment comprising an oligonucleotide sequenceconjugated at the 3′-position, an oligonucleotide sequence conjugated atthe 5′-position, linear oligonucleotide sequences, branchedoligonucleotide sequences, LNAs, PNAs, oligonucleotide sequencesoptionally covalently attached to other moieties, or combinations orderivatives thereof.

Example 41C

The method of any one of Examples 1C-40C, wherein a plurality ofmolecular probes and a plurality of particles, beads, or other surfaces,are provided to the sample.

Example 42C

The method of any one of Examples 1C-41C, wherein a plurality ofuniversal adapters are provided to the sample.

Example 43C

The method of any one of Examples 1C-42C, wherein the binding affinityfor the at least first target is 10⁻⁴ M or less.

Example 44C

The method of any one of Examples 1C-43C, wherein the method comprisesan automated system or robotic system.

Example 45C

The method of any one of Examples 1C-44C, wherein the other surfacecomprises a scaffold, a plate, or solid array.

Example 46C

The method of any one of Examples 1C-44C, wherein the scaffold comprisesa dendrimer, a polysaccharide, a dextran, a protein, a peptide, afurther oligonucleotide sequence, a portion of the secondoligonucleotide sequence that is not complementary to the firstoligonucleotide sequence of the molecular probe, a polymer, ahydrophilic polymer, a bead, a nanoparticle, or combinations orderivatives thereof.

Example 1D

A method for assaying a target of a sample, comprising: i) providing tothe sample: 1) a first molecular probe, comprising a first bindingmoiety conjugated to a first oligonucleotide sequence; and 2) a firstbead conjugate, comprising a first bead conjugated to a secondoligonucleotide sequence that is complementary to the firstoligonucleotide sequence of the first molecular probe, wherein the firstbead comprises or is encoded with one or more signal generatingmoieties; ii) binding the target in the sample with the first bindingmoiety of the first molecular probe; iii) hybridizing the firstoligonucleotide sequence of the first molecular probe with the secondoligonucleotide sequence of the first bead conjugate; and iv) providingto the sample a second binding moiety comprising one or more signalgenerating moieties; v) further binding the target of the first bindingmoiety-bound target with the second binding moiety to form asandwich-complex; vi) detecting a signal generated from the sandwichcomplex;

wherein the method is characterized by one or more of the following: a)the conjugation between the first oligonucleotide sequence and the firstbinding moiety and conjugation between the complementary secondoligonucleotide sequence and the first bead conjugate, comprises one ormore covalent bond linkages, comprising a hydrazone, oxime, triazine, orother covalent bond, wherein the formation of the conjugates are atleast 90% efficient; and b) the first binding moiety and the secondbinding moiety comprise strong binding affinities for the target.

Example 2D

The method of Example 1D, wherein the mode of addition comprises: i) thefirst molecular probe and the first bead conjugate are combined togetherand hybridized prior to contacting the sample; ii) the first molecularprobe is combined with the sample prior to the addition of the firstbead conjugate; or iii) the first bead conjugate is combined with thesample prior to the addition of the first molecular probe.

Example 3D

The method of any one of Examples 1D-2D, wherein the method comprises:i) the first molecular probe binding the target prior to hybridizingwith the first bead conjugate; or ii) the first molecular probehybridizing with the first bead conjugate prior to binding the target.

Example 4D

A method for assaying a target of a sample, comprising: i) providing tothe sample: 1) a first molecular probe, comprising a first bindingmoiety conjugated to a first oligonucleotide sequence; 2) a first beadconjugate, comprising a first bead conjugated to a secondoligonucleotide sequence, wherein the first bead comprises or is encodedwith one or more signal generating moieties; and 3) a first universaladapter, comprising an oligonucleotide sequence having a first sequencesegment complementary to the first oligonucleotide sequence of the firstmolecular probe and a second sequence segment complementary to thesecond oligonucleotide sequence of the first bead conjugate; ii) bindingthe target in the sample with the first binding moiety of the firstmolecular probe; iii) hybridizing the first oligonucleotide sequence ofthe first molecular probe to the first oligonucleotide sequence segmentof the first universal adapter; iv) hybridizing the secondoligonucleotide sequence of the first bead conjugate to thecomplementary second oligonucleotide sequence segment of the firstuniversal adapter; and v) providing to the sample a second bindingmoiety comprising one or more signal generating moieties; iv) furtherbinding the target of the first binding moiety-bound target with thesecond binding moiety to form a sandwich-complex; vi) detecting a signalgenerated from the sandwich complex; wherein the method is characterizedby one or more of the following: a) the conjugation between the firstoligonucleotide sequence and the first binding moiety and conjugationbetween the complementary second oligonucleotide sequence and the firstbead conjugate, comprises one or more covalent bond linkages, comprisinga hydrazone, oxime, triazine, or other covalent bond, wherein theformation of the conjugates are at least 90% efficient; and b) the firstbinding moiety and the second binding moiety comprise strong bindingaffinities for the target.

Example 5D

The method of Example 4D, wherein the mode of addition comprises: i) thefirst molecular probe, the first universal adapter, and the first beadconjugate are combined together and hybridized prior to contacting thesample; ii) the first molecular probe and the first universal adapterare combined together and hybridized prior to contacting the sample;iii) the first bead conjugate and the first universal adapter arecombined together and hybridized prior to contacting the sample; iv) thefirst molecular probe, alone or in combination with the first beadconjugate, is combined with the sample prior to the addition of thefirst universal adapter; or v) the first universal adapter is combinedwith the sample prior to the addition of the first molecular probeand/or the first bead conjugate.

Example 6D

The method of Example 4D or 5D, wherein the method comprises: i) thefirst molecular probe hybridizing with the first universal adapter priorto said first molecular probe binding the target; ii) the firstmolecular probe hybridizing with the first universal adapter after saidfirst molecular probe binds the target; iii) the first bead conjugatehybridizing with the first universal adapter prior to the firstmolecular probe binding the target; iv) the first bead conjugatehybridizing with the first universal adapter after the first molecularprobe binds the target; v) the first universal adapter hybridizing withthe first molecular probe and hybridizing with the first bead conjugateprior to said first molecular probe binding the target; or vi) the firstuniversal adapter hybridizing with the first molecular probe andhybridizing with the first bead conjugate after said first molecularprobe binds the target.

Example 7D

A method for crosslinking, comprising: i) introducing to a samplecomprising one or more targets: a) one or more firstantibody-oligonucleotide conjugates, comprising a first antibodyconjugated to a first oligonucleotide sequence; and b) one or moresecond antibody-oligonucleotide conjugates, comprising a second antibodyconjugated to a second oligonucleotide sequence; ii) binding at least afirst target of the one or more targets with the first antibody of theone or more first antibody-oligonucleotide conjugates and with thesecond antibody of the one or more second antibody-oligonucleotideconjugates to form one or more sandwich-complexes; iii) contacting theone or more sandwich-complexes with: a) one or more firstbead-oligonucleotide conjugate, comprising a first bead conjugated to acomplementary first oligonucleotide sequence; and b) one or more secondbead-oligonucleotide conjugate, comprising a second bead conjugated to acomplementary second oligonucleotide sequence; iv) crosslinking the oneor more sandwich-complexes by: a) hybridizing the first oligonucleotidesequences of the one or more sandwich-complexes with the complementaryfirst oligonucleotide sequences of the one or more firstbead-oligonucleotide conjugates; and b) hybridizing the secondoligonucleotide sequences of the one or more sandwich-complexes with thecomplementary second oligonucleotide sequences of the one or more secondbead-oligonucleotide conjugates.

Example 8D

The crosslinking method of Example 7D, wherein the formation of thecrosslinked one or more sandwich-complexes forms an agglutination.

Example 9D

The crosslinking method of Examples 7D or 8D, wherein the method furthercomprises detecting, measuring, and/or quantifying the degree of theformed agglutination to determine the amount of the one or more targetsin the sample.

Example 10D

The crosslinking method of any one of Examples 7D-9D, wherein the firstantibody or the second antibody comprise a monoclonal antibody or apolyclonal antibody.

Example 11D

The crosslinking method of any one of Examples 7D-10, wherein: i) thefirst antibody comprises a first polyclonal antibody and the secondantibody comprises a second polyclonal antibody; ii) the first antibodycomprises a first monoclonal antibody and the second antibody comprisesa second monoclonal antibody; iii) the first antibody comprises a firstmonoclonal antibody and the second antibody comprises a first polyclonalantibody; or iv) the first antibody comprises a first polyclonalantibody and the second antibody comprises a first monoclonal antibody.

Example 12D

The crosslinking method of Example 11D, wherein the first monoclonalantibody is raised against a first epitope of the target and the secondmonoclonal antibody is raised against a second epitope of the target.

Example 13D

The crosslinking method of any one of Examples 7D-12D, wherein the firstantibody comprises a first polyclonal antibody and the second antibodycomprises a second polyclonal antibody.

Example 14D

The crosslinking method of Example 13D, wherein a first portion of thefirst polyclonal antibody binds to a first epitope of the target and asecond portion of the second polyclonal antibody binds to a secondepitope of the target.

Example 15D

The crosslinking method of any one of Examples 7D-12D, wherein the firstantibody comprises a first monoclonal antibody and the second antibodycomprises a second monoclonal antibody.

Example 16D

The crosslinking method of any one of Examples 7D-12D, wherein the firstantibody comprises a first monoclonal antibody and the second antibodycomprises a first polyclonal antibody.

Example 17D

The method of any one of Examples 1D-16D, wherein: i) the assaycomprises a singleplex or multiplex assay; and ii) the assay detects,measures, or quantifies the level of binding and/or amount of the targetpresent in the sample.

Example 18D

The method of any one of Examples 1D-17D, wherein method detects,measures, or quantifies the level of binding and/or amount of the targetpresent in the sample with one or more of the following:immunoturbidity, latex agglutination, gold particle agglutination,visual inspection, a change in light transmittance through said sample,increased light transmittance through said sample, flow cytometry,immunomagnetic cellular depletion, immunomagnetic cell capture, array,bead array, multiplex bead array, microarray, antibody array, cellulararray, chemiluminescence, infrared, microscopy, imaging, high contentscreening (HCS), mass cytometry, lateral flow immunoassay,immunodetection, immunohistochemistry (IHC), immunocytochemistry (ICC),in situ hybridization (ISH), enzyme immuno-assay (EIA), enzyme linkedimmuno-assay (ELISA), ELISpot, a blotting method, a Western blot, aSouthern blot, a Southwestern blot, labeling inside an electrophoresissystem, labeling on a surface, labeling on an array, PCR amplification,elongation followed by PCR amplification, immunoprecipitation,co-immunoprecipitation, chromatin immunoprecipitation, pretargetingimaging, therapeutic agent, or combinations thereof.

Example 19D

The method of any one of Examples 1D-18D, wherein: i) the assaycomprises a singleplex or multiplex assay; and ii) the assay detects,measures, or quantifies the level of binding and/or amount of the targetpresent in the sample with one or more of the following:immunoturbidity, latex agglutination, gold particle agglutination,visual inspection, a change in light transmittance through said sample,increased light transmittance through said sample, lateral flowimmunoassay, immunodetection, flow cytometry, microscopy, imaging, highcontent screening (HCS), multiplex bead array, microarray, antibodyarray, cellular array, immunohistochemistry (IHC), immunocytochemistry(ICC), in situ hybridization (ISH), enzyme immuno-assay (EIA), enzymelinked immuno-assay (ELISA), ELISpot, or a blotting method.

Example 20D

The method of any one of Examples 1D-19D, wherein: i) the assaycomprises a singleplex or multiplex assay; and ii) the assay detects,measures, or quantifies the level of binding and/or amount of the targetpresent in the sample with one or more of the following:immunoturbidity, latex agglutination, gold particle agglutination,visual inspection, a change in light transmittance through said sample,increased light transmittance through said sample, lateral flowimmunoassay, immunodetection, flow cytometry, microscopy, imaging, highcontent screening (HCS), mass cytometry, lateral flow immunoassay,immunohistochemistry (IHC), immunocytochemistry (ICC),immunoprecipitation, pretargeting imaging, therapeutic agent, orcombinations thereof.

Example 21D

The method of any one of Examples 1D-20D, wherein the sample ischaracterized as at least one or more of the following: i) a complexsample; and ii) a homogeneous or a heterogeneous mixture; wherein saidsample comprises at least one or more of the following: a) one or moreanalytes having substantially the same or substantially differentbinding specificities; b) one or more of the following biologiccomponents, comprising: cells, membranes, biological molecules,metabolites, or disease biomarkers; and c) a biological fluid or afluidized biological tissue.

Example 22D

The method of any one of Examples 1D-21D, wherein the hybridizationefficiency of the first oligonucleotide sequence to the secondoligonucleotide sequence is at least 50% with respect to the first bead,under the hybridization conditions employed.

Example 23D

The method of any one of Examples 1D-22D, wherein the first molecularprobe comprises one or more of the following properties: i) a molecularweight of between about 15,000 Daltons to about 450,000 Daltons; ii) asolubility that is substantially the same as that of the unconjugatedfirst binding moiety; iii) a solubility that minimizes non-specificbinding to the target; iv) the first oligonucleotide sequence of thefirst molecular probe does not adversely affect the solubility of thefirst binding moiety; v) interacts and binds to the target viainteractions other than exclusively electrostatic; vi) a unique,distinguishable, and/or specifically designed first oligonucleotidesequence; and vii) the first oligonucleotide sequence of the firstmolecular probe is uniquely and specifically designed to hybridize tothe second oligonucleotide sequence of the first bead.

Example 24D

The method of any one of Examples 1D-23D, wherein the method ofdetection generates less false positives than secondary antibodydetection methods.

Example 25D

The method of any one of Examples 1D-24D, wherein the method furthercomprises: i) preparing the first molecular probe; ii) preparing thefirst bead conjugate; and iii) optionally preparing the first universaladapter; wherein the prepared first molecular probe, prepared first beadconjugate, and optionally prepared first universal adapter, have atleast 90% purity.

Example 26D

The method of any one of Examples 1D-25D, wherein the method furthercomprises preparing and isolating the first molecular probe, comprising:i) providing the first binding moiety; ii) conjugating the first bindingmoiety with at least one first oligonucleotide sequence at greater than90% efficiency to form first binding moiety-oligonucleotide conjugates;and iii) isolating the first binding moiety-oligonucleotide conjugatesfrom the conjugation mixture by binding, retaining, and/or retarding asubstantial portion of: a) the conjugates, removing a substantialportion of the unconjugated first oligonucleotide sequence in a washstep followed by release of the bound, retained, and/or retardedconjugates; or b) the unconjugated first oligonucleotide sequences,followed by collecting a substantial portion of the non-bound,non-retained, and/or non-retarded conjugates in a wash step.

Example 27D

The method of any one of Examples 1D-26D, wherein the method furthercomprises preparing and isolating the first bead conjugate, comprising:i) providing the first bead; ii) conjugating the second oligonucleotidesequence with the first bead at greater than 90% efficiency to formfirst bead-second oligonucleotide conjugates; and iii) isolating thefirst bead-second oligonucleotide conjugates from the conjugationmixture by binding, retaining, and/or retarded a substantial portion of:a) the conjugates, removing a substantial portion of the unconjugatedsecond oligonucleotide sequences in a wash step followed by release ofthe bound, retained, and/or retarded conjugates; or b) the unconjugatedsecond oligonucleotide sequences, followed by collecting a substantialportion of the non-bound, non-retained, and/or non-retarded conjugatesin a wash step.

Example 28D

The method of any one of Examples 1D-27D, wherein the first beadconjugate further comprises a scaffold conjugated to the secondoligonucleotide sequence, and wherein said scaffold comprises one ormore signal generating moieties.

Example 29D

The method of Example 28D, wherein the scaffold comprises a dendrimer, apolysaccharide, a dextran, a protein, a peptide, a furtheroligonucleotide sequence, a portion of the second oligonucleotidesequence that is not complementary to the first oligonucleotide sequenceof the molecular probe, a polymer, a hydrophilic polymer, a bead, ananoparticle, or combinations or derivatives thereof.

Example 30D

The method of any one of Examples 1D-29D, wherein the method furthercomprises preparing and isolating the first bead conjugate furthercomprising a scaffold conjugated to the second oligonucleotide sequence,wherein the scaffold comprises one or more signal generating moieties,said method comprising: i) providing a plurality of the scaffoldscomprising the one or more signal generating moieties; ii) conjugatingthe second oligonucleotide sequence with at least one of the pluralityof scaffolds at greater than 90% efficiency to form scaffold-secondoligonucleotide conjugates; and iii) isolating the scaffold-secondoligonucleotide conjugates from the conjugation mixture by binding,retaining, and/or retarding a substantial portion of: a) the conjugates,removing a substantial portion of the unconjugated secondoligonucleotide sequences in a wash step followed by release of thebound, retained, and/or retarded conjugates; or b) the unconjugatedsecond oligonucleotide sequences, followed by collecting a substantialportion of the non-bound, non-retained, and/or non-retarded conjugatesin a wash step.

Example 31D

The method of any one of Examples 26D-30D, wherein the isolation steputilizes an immobilized binder, chromatography, affinity chromatography,size exclusion chromatography, HPLC, reverse-phase chromatography,electrophoresis, capillary electrophoresis, polyacrylamide gelelectrophoresis, agarose gel electrophoresis, free flow electrophoresis,differential centrifugation, thin layer chromatography,immunoprecipitation, hybridization, solvent extraction, dialysis,filtration, diafiltration, tangential flow filtration, ion exchangechromatography, hydrophobic interaction chromatography, or combinationsthereof.

Example 32D

The method of any one of Examples 26D-30D, wherein the isolation steputilizes an immobilized binder, affinity chromatography, size exclusionchromatography, electrophoresis, differential centrifugation,immunoprecipitation, hybridization, solvent extraction, dialysis,filtration, diafiltration, ion exchange chromatography, hydrophobicinteraction chromatography, or combinations thereof.

Example 33D

The method of any one of Examples 26D-30D, wherein the isolation steputilizes an immobilized binder, affinity chromatography, size exclusionchromatography, differential centrifugation, dialysis, filtration,hydrophobic interaction chromatography, or combinations thereof.

Example 34D

The method of any one of Examples 1D-33D, wherein the binding moietycomprises an antibody, a monoclonal antibody, a polyclonal antibody, anenzyme, a protein, a peptide, a carbohydrate, a nuclear receptor, asmall molecule, an aptamer, a chelator, or combinations or derivativesthereof.

Example 35D

The method of any one of Examples 1D-34D, wherein the sample comprisesone or more targets.

Example 36D

The method of any one of Examples 1D-35D, wherein the target is abiological target.

Example 37D

The method of Example 36D, wherein the biological target comprises anantigen, a pathogen, a protein, a peptide, an epitope, acarbohydrate-containing molecule, a small molecule, or combinations orderivatives thereof.

Example 38D

The method of any one of Examples 1D-37D, wherein the signal generatingmoiety or the one or more signal generating moieties of the first beadconjugate, the hybridized first bead conjugate, the second bindingmoiety, or of the scaffold, comprises one or more of the following: adirectly detectable signal generating moiety, an indirectly detectablesignal generating moiety, a fluorescent dye, a fluorophore, afluorochrome, a chromophore, a biofluorescent protein, a luminescentspecies, a chemiluminescent compound, a electrochemiluminescent label, abioluminescent label, a phosphorescent species, a fluorophore labeledDNA dendrimer, Quantum Dot, a tandem dye, a FRET dye, a heavy atom, aspin label, a radioactive isotope, a nanoparticle, a light scatteringnanoparticle or microsphere, a diffracting particle, a polymer, apolymer particle, a bead, a solid surface, a Raman particle, a metalparticle, a stable isotope, a heavy metal chelate, a magnetic particle,an RFID tag, a microbarcode particle, an enzyme, an enzyme substrate, amolecule specifically recognized by another substance carrying a labelor reacts with a substance carrying a label, an antibody, an antibodyfragment, an antigen, a nucleic acid, a nucleic acid analog,oligonucleotide, oligonucleotide analog, complementary oligonucleotide,complementary oligonucleotide analog, a ligand, a protein, a peptideligand, a protein substrate, a receptor; a substrate, a secondaryreporter, a hapten, or combinations or derivatives thereof.

Example 39D

The method of any one of Examples 1D-38D, wherein the one or more signalgenerating moieties provides an enhanced signal that minimizes detectionerrors from background noise, relative to conventionally labeled bindingmoieties.

Example 40D

The method of any one of Examples 1D-39D, wherein the molecular probe,the first bead conjugate, and/or universal adapter further comprises aspacer group, comprising a polymerized ethylene oxide, a PEG, a PEO, aprotein, a peptide, a DNA, an RNA, an oligonucleotide sequence, or adextran.

Example 41D

The method of any one of Examples 1D-40D, wherein the binding moiety,the first bead, the scaffold, the first oligonucleotide sequence, and/orthe second oligonucleotide sequence comprise HyNic or 4-FB.

Example 42D

The method of any one of Examples 1D-41D, wherein the first beadconjugate comprises a unique, distinguishable, and/or specificallydesigned second oligonucleotide sequence.

Example 43D

The method of any one of Examples 1D-42D, wherein the firstoligonucleotide sequence, second oligonucleotide sequence, and/oroligonucleotide sequence segment comprising an oligonucleotide sequenceconjugated at the 3′-position, an oligonucleotide sequence conjugated atthe 5′-position, linear oligonucleotide sequences, branchedoligonucleotide sequences, LNAs, PNAs, oligonucleotide sequencesoptionally covalently attached to other moieties, or combinations orderivatives thereof.

Example 44D

The method of any one of Examples 1D-43D, wherein the sample comprisesone or more targets.

Example 45D

The method of any one of Examples 1D-44D, wherein a plurality ofmolecular probes and a plurality of bead conjugates are provided to thesample.

Example 46D

The method of any one of Examples 1D-45D, wherein a plurality ofuniversal adapters are provided to the sample.

Example 47D

The method of any one of Examples 1D-46D, wherein the binding affinityof the first binding moiety and/or of the second binding moiety for thetarget is 10⁻⁴ M or less.

Example 48D

The method of any one of Examples 1D-47D, wherein the binding affinityof the first binding moiety and/or of the second binding moiety for theat least first target is 10⁻⁴ M or less.

Example 49D

The method of any one of Examples 1D-48D, wherein the binding affinityof the first binding moiety and/or of the second binding moiety for theat least second target is 10⁻⁴ M or less.

Example 50D

The method of any one of Examples 1D-49D, wherein the method comprisesan automated system or robotic system.

Example 51D

The method of any one of Examples 1D-50D, wherein the first beadconjugate comprises or is encoded with one or more of the following: adirectly detectable signal generating moiety, an indirectly detectablesignal generating moiety, a fluorescent dye, a fluorophore, afluorochrome, a chromophore, a biofluorescent protein, a luminescentspecies, a chemiluminescent compound, a electrochemiluminescent label, abioluminescent label, a phosphorescent species, a fluorophore labeledDNA dendrimer, Quantum Dot, a tandem dye, a FRET dye, a heavy atom, aspin label, a radioactive isotope, a nanoparticle, a light scatteringnanoparticle or microsphere, a diffracting particle, a polymer, apolymer particle, a bead, a solid surface, a Raman particle, a metalparticle, a stable isotope, a heavy metal chelate, a magnetic particle,an RFID tag, a microbarcode particle, an enzyme, an enzyme substrate, amolecule specifically recognized by another substance carrying a labelor reacts with a substance carrying a label, an antibody, an antibodyfragment, an antigen, a nucleic acid, a nucleic acid analog,oligonucleotide, oligonucleotide analog, complementary oligonucleotide,complementary oligonucleotide analog, a ligand, a protein, a peptideligand, a protein substrate, a receptor; a substrate, a secondaryreporter, a hapten, or combinations or derivatives thereof.

Example 52D

The method of any one of Examples 1D-50D, wherein the first beadconjugate comprises or is encoded with one or more of the following: afluorescent dye, a fluorophore, a fluorochrome, a fluorescent protein, abiofluorescent protein, a luminescent species, a chemiluminescentcompound, a electrochemiluminescent label, a fluorophore labeled DNAdendrimer, Quantum Dot, a secondary reporter, a hapten, an enzyme, anantibody, a nanoparticle, a light scattering nanoparticle ormicrosphere, a bioluminescent label, a tandem dye, a FRET dye, adiffracting particle, a polymer particle, a bead, a solid surface, ametal particle, a molecule specifically recognized by another substancecarrying a label or reacts with a substance carrying a label, a nucleicacid, a nucleic acid analog, oligonucleotide, oligonucleotide analog,complementary oligonucleotide, complementary oligonucleotide analog.

Example 53D

A method for assaying one or more targets of a sample, comprising: i)providing to the sample: 1) a plurality of molecular probes, comprising:A) at least a first molecular probe having a first binding moietyconjugated to a first oligonucleotide sequence; and B) at least a secondmolecular probe having a second binding moiety conjugated to a secondoligonucleotide sequence; and 2) a plurality of detectable components,comprising: A) at least a first detectable component, comprising a firstbead conjugated to a complementary first oligonucleotide sequence,wherein the first bead comprises one or more signal generating moieties;B) at least a second detectable component, comprising a second beadconjugated to a complementary second oligonucleotide sequence, whereinthe second bead comprises one or more signal generating moieties; ii)binding the one or more targets, comprising at least one of thefollowing: 1) binding at least a first target of the one or more targetsin the sample with the first binding moiety of the at least firstmolecular probe; and 2) binding at least a second target of the one ormore targets in the sample with the second binding moiety of the atleast second molecular probe; iii) hybridizing the plurality ofmolecular probes and the plurality of detectable components, comprisingat least one of the following: 1) hybridizing the first oligonucleotidesequence of at least first molecular probe to the first complementaryoligonucleotide sequence segment of the at least first detectablecomponent; and 2) hybridizing the second oligonucleotide sequence of atleast second molecular probe to the second complementary oligonucleotidesequence segment of the at least second detectable component; and iv)detecting one or more signals generated from at least one of thefollowing: 1) the at least first hybridized detectable component; and 2)the at least second hybridized detectable component; wherein the methodis characterized by one or more of the following: a) the conjugationbetween the first oligonucleotide sequence and the first binding moiety,between the second oligonucleotide sequence and the second bindingmoiety, between the first complementary oligonucleotide sequence and thefirst bead, and between the second complementary oligonucleotidesequence and the second bead, comprises one or more covalent bondlinkages, comprising a hydrazone, oxime, triazine, or other covalentbond, wherein the formation of the conjugates are at least 90%efficient; and b) the first binding moiety comprises a strong bindingaffinity for the at least first target of the one or more targets andthe second binding moiety comprises a strong binding affinity for the atleast second target of the one or more targets.

Example 54D

A method for assaying one or more targets of a sample, comprising: i)providing to the sample: a) a plurality of molecular probes, comprising:a first oligonucleotide sequence independently paired, via conjugation,to a plurality of binding moieties comprising at least a first bindingmoiety and at least a second binding moiety; b) a plurality ofdetectable components, comprising: a plurality of second oligonucleotidesequences independently paired, via conjugation, to a plurality of beadshaving one or more signal generating moieties comprising at least afirst bead and at least a second bead; and c) a plurality of universaladapters, comprising: a first oligonucleotide sequence segment,complementary to the first oligonucleotide sequence of said plurality ofmolecular probes, independently paired with a plurality of secondoligonucleotide sequence segments complementary to the plurality ofsecond oligonucleotide sequences of said plurality of detectablecomponents; ii) binding the one or more targets, comprising at least oneof the following: a) binding at least a first target of the one or moretargets in the sample with the first binding moiety of the at leastfirst molecular probe; and b) binding at least a second target of theone or more targets in the sample with the second binding moiety of theat least second molecular probe; iii) hybridizing the plurality ofmolecular probes and the plurality of detectable components with theplurality of universal adapters; and iv) detecting one or more signalsgenerated from at least one of the following: a) the at least firsthybridized detectable component; and b) the at least second hybridizeddetectable component; wherein the method is characterized by one or moreof the following: A) the conjugation between the first oligonucleotidesequence and the first binding moiety, between the secondoligonucleotide sequence and the second binding moiety, between thefirst complementary oligonucleotide sequence and the first bead, andbetween the second complementary oligonucleotide sequence and the secondbead, comprises one or more covalent bond linkages, comprising ahydrazone, oxime, triazine, or other covalent bond, wherein theformation of the conjugates are at least 90% efficient; and B) the firstbinding moiety comprises a strong binding affinity for the at leastfirst target of the one or more targets and the second binding moietycomprises a strong binding affinity for the at least second target ofthe one or more targets.

Example 1E

A tunable detection system, comprising: i) a molecular probe prepared byconjugating a first oligonucleotide sequence to a binding moiety; andii) a series of detectable components, comprising a range of signalgenerating moieties conjugated to a second oligonucleotide sequence,wherein the range of signal generating moieties generates a range ofsignal intensities, and wherein the second oligonucleotide sequence iscomplementary to the first oligonucleotide sequence; wherein the rangeof signal intensities generated can be tuned over a range from the limitof self-quenching to the intensity of a single signal generating moiety.

Example 2E

A tunable detection system, comprising: i) a molecular probe prepared byconjugating a first oligonucleotide sequence to a binding moiety; andii) a series of detectable components, comprising a range of signalgenerating moieties conjugated to a second oligonucleotide sequence,wherein the range of signal generating moieties generates a range ofsignal intensities; and iii) a universal adapter, comprising a firstoligonucleotide sequence segment complementary to the firstoligonucleotide sequence and a second oligonucleotide sequence segmentcomplementary to the second oligonucleotide sequence; wherein the rangeof signal intensities generated can be tuned over a range from the limitof self-quenching to intensity of a single signal generating moiety.

Example 3E

The tunable detection system of Examples 1E or 2E, wherein the signalgenerated is from a target in a sample bound by the molecular probe thatis hybridized to the detectable component.

Example 4E

The tunable detection system of any one of Examples 1E-3E, wherein thedetectable component or the hybridized detectable component comprisesone or more signal generating moieties, comprising one or more of thefollowing: a directly detectable signal generating moiety, an indirectlydetectable signal generating moiety, a fluorescent dye, a fluorophore, afluorochrome, a chromophore, a biofluorescent protein, a luminescentspecies, a chemiluminescent compound, a electrochemiluminescent label, abioluminescent label, a phosphorescent species, a fluorophore labeledDNA dendrimer, Quantum Dot, a tandem dye, a FRET dye, a heavy atom, aspin label, a radioactive isotope, a nanoparticle, a light scatteringnanoparticle or microsphere, a diffracting particle, a polymer, apolymer particle, a bead, a solid surface, a Raman particle, a metalparticle, a stable isotope, a heavy metal chelate, a magnetic particle,an RFID tag, a microbarcode particle, an enzyme, an enzyme substrate, amolecule specifically recognized by another substance carrying a labelor reacts with a substance carrying a label, an antibody, an antibodyfragment, an antigen, a nucleic acid, a nucleic acid analog,oligonucleotide, oligonucleotide analog, complementary oligonucleotide,complementary oligonucleotide analog, a ligand, a protein, a peptideligand, a protein substrate, a receptor; a substrate, a secondaryreporter, a hapten, or combinations thereof.

Example 5E

The tunable detection system of any one of Examples 1E-4E, wherein thedetectable component comprises a scaffold conjugated to the secondoligonucleotide sequence, and wherein said scaffold comprises the one ormore signal generating moieties.

Example 6E

The tunable detection system of any one of Examples 1E-5E, wherein thescaffold comprises a dendrimer, a polysaccharide molecule, a dextran, aprotein, a peptide, a second oligonucleotide sequence, a portion of theoligonucleotide sequence that is not complementary to theoligonucleotide sequence of the molecular probe, a polymer, ahydrophilic polymer, a bead, a nanoparticle, or combinations orderivatives thereof.

Example 7E

The tunable detection system of any one of Examples 1E-6E, wherein thetuning of the signal intensities generated is determined by selectingthe identity of the detectable component or by having a greater orlesser number of signal generating moieties.

Example 8E

The tunable detection system of any one of Examples 1E-7E, wherein: i)the tunable detection system comprises a singleplex or multiplex tunabledetection system; and ii) the tunable detection system detects,measures, or quantifies the level of binding and/or amount of one ormore targets present in a sample from the generated signal by one ormore of the following: flow cytometry, immunomagnetic cellulardepletion, immunomagnetic cell capture, array, bead array, multiplexbead array, microarray, antibody array, cellular array,chemiluminescence, infrared, microscopy, imaging, high content screening(HCS), mass cytometry, lateral flow immunoassay, immunodetection,immunohistochemistry (IHC), immunocytochemistry (ICC), in situhybridization (ISH), enzyme immuno-assay (EIA), enzyme linkedimmuno-assay (ELISA), ELISpot, immunoturbidity, latex agglutination,gold particle agglutination, visual inspection, a change in lighttransmittance through said sample, increased light transmittance throughsaid sample, a blotting method, a Western blot, a Southern blot, aSouthwestern blot, labeling inside an electrophoresis system, labelingon a surface, labeling on an array, PCR amplification, elongationfollowed by PCR amplification, immunoprecipitation,co-immunoprecipitation, chromatin immunoprecipitation, pretargetingimaging, therapeutic agent, or combinations thereof.

Example 9E

The tunable detection system of any one of Examples 1E-8E, wherein thetunable detection system minimizes signal spillover.

Example 10E

A tunable detection system, comprising: i) a plurality of molecularprobes comprising: 1) at least a first molecular probe prepared byconjugating a first oligonucleotide sequence to a first binding moiety;and 2) at least a second molecular probe prepared by conjugating asecond oligonucleotide sequence to a second binding moiety; and ii) aplurality of detectable components comprising: 1) at least a firstdetectable component comprising a range of first signal generatingmoieties conjugated to a oligonucleotide sequence complementary to saidfirst oligonucleotide sequence, wherein the range of first signalgenerating moieties generates a range of signal intensities; and 2) atleast a second detectable component comprising a range of second signalgenerating moieties conjugated to a oligonucleotide sequencecomplementary to said second oligonucleotide sequence, wherein the rangeof second signal generating moieties generates a range of signalintensities; wherein the range of signal intensities generated from theat least first detectable component and the at least second detectablecomponent can be individually tuned over a range from the limit ofself-quenching to intensity of the single first signal generating moietyor the second signal generating moiety, respectively.

Example 11E

A tunable detection system, comprising: i) a plurality of molecularprobes comprising a plurality of binding moieties independentlyconjugated to a universal oligonucleotide sequence, comprising: 1) atleast a first molecular probe comprising a first binding moietyconjugated to a universal oligonucleotide sequence; and 2) at least asecond molecular probe comprising a second binding moiety conjugated toa universal oligonucleotide sequence; and ii) a plurality of detectablecomponents comprising a range of first signal generating moietiesindependently conjugated to a plurality of oligonucleotide sequences,comprising: 1) at least a first detectable component comprising a rangeof first signal generating moieties conjugated to first oligonucleotidesequence, wherein the range of first signal generating moietiesgenerates a range of signal intensities; and 2) at least a seconddetectable component comprising a range of second signal generatingmoieties conjugated to a second oligonucleotide sequence,

wherein the range of second signal generating moieties generates a rangeof signal intensities; and iii) a plurality of universal adapters,comprising a first oligonucleotide sequence segment complementary to theuniversal oligonucleotide sequence independently paired with a pluralityof oligonucleotide sequence segments complementary to the plurality ofoligonucleotide sequence of the plurality of detectable components;wherein the range of signal intensities generated from the at leastfirst detectable component and the at least second detectable componentcan be individually tuned over a range from the limit of self-quenchingto intensity of the single first signal generating moiety or the secondsignal generating moiety, respectively.

Example 12E

The tunable detection system of Examples 10E or 11E, wherein: i) thefirst signal generated is from at least a first target in a sample boundby the at least first molecular probe that is hybridized to the at leastfirst detectable component; and ii) the second signal generated is fromat least a second target in the sample bound by the at least secondmolecular probe that is hybridized to the at least second detectablecomponent.

Example 13E

The tunable detection system of any one of Examples 10E-12E, wherein theplurality of detectable components, the at least first hybridizeddetectable component, and/or the at least second hybridized detectablecomponent comprise one or more signal generating moieties, wherein saidone or more signal generating moieties comprises one or more of thefollowing: a directly detectable signal generating moiety, an indirectlydetectable signal generating moiety, a fluorescent dye, a fluorophore, afluorochrome, a chromophore, a biofluorescent protein, a luminescentspecies, a chemiluminescent compound, a electrochemiluminescent label, abioluminescent label, a phosphorescent species, a fluorophore labeledDNA dendrimer, Quantum Dot, a tandem dye, a FRET dye, a heavy atom, aspin label, a radioactive isotope, a nanoparticle, a light scatteringnanoparticle or microsphere, a diffracting particle, a polymer, apolymer particle, a bead, a solid surface, a Raman particle, a metalparticle, a stable isotope, a heavy metal chelate, a magnetic particle,an RFID tag, a microbarcode particle, an enzyme, an enzyme substrate, amolecule specifically recognized by another substance carrying a labelor reacts with a substance carrying a label, an antibody, an antibodyfragment, an antigen, a nucleic acid, a nucleic acid analog,oligonucleotide, oligonucleotide analog, complementary oligonucleotide,complementary oligonucleotide analog, a ligand, a protein, a peptideligand, a protein substrate, a receptor; a substrate, a secondaryreporter, a hapten, or combinations thereof.

Example 14E

The tunable detection system of any one of Examples 10E-13E, wherein theat least first hybridized detectable component comprises a firstscaffold conjugated to the first oligonucleotide sequence and/or the atleast second hybridized detectable component comprises a second scaffoldconjugated to the second oligonucleotide sequence.

Example 15E

The tunable detection system of any one of Examples 10E-14E, wherein thefirst scaffold and/or the second scaffold comprises a dendrimer, apolysaccharide molecule, a dextran, a protein, a peptide, an additionaloligonucleotide sequence, a portion of the first or secondoligonucleotide sequence that is not complementary to the first orsecond oligonucleotide sequence of the at least first or secondmolecular probe, a polymer, a hydrophilic polymer, a bead, ananoparticle, or combinations or derivatives thereof.

Example 16E

The tunable detection system of any one of Examples 10E-15E, wherein thefirst scaffold and/or the second scaffold has one or more signalgenerating moieties.

Example 17E

The tunable detection system of any one of Examples 10E-16E, wherein: i)the tunable detection system comprises a singleplex or multiplex tunabledetection system; and ii) the tunable detection system detects,measures, or quantifies the level of binding and/or amount of one ormore targets present in a sample from the signal generated from the atleast first detectable component and/or the signal generated from the atleast second detectable component by one or more of the following: flowcytometry, immunomagnetic cellular depletion, immunomagnetic cellcapture, array, bead array, multiplex bead array, microarray, antibodyarray, cellular array, chemiluminescence, infrared, microscopy, imaging,high content screening (HCS), mass cytometry, lateral flow immunoassay,immunodetection, immunohistochemistry (IHC), immunocytochemistry (ICC),in situ hybridization (ISH), enzyme immuno-assay (EIA), enzyme linkedimmuno-assay (ELISA), ELISpot, immunoturbidity, latex agglutination,gold particle agglutination, visual inspection, a change in lighttransmittance through said sample, increased light transmittance throughsaid sample, a blotting method, a Western blot, a Southern blot, aSouthwestern blot, labeling inside an electrophoresis system, labelingon a surface, labeling on an array, PCR amplification, elongationfollowed by PCR amplification, immunoprecipitation,co-immunoprecipitation, chromatin immunoprecipitation, pretargetingimaging, therapeutic agent, or combinations thereof.

Example 18E

The tunable detection system of any one of Examples 10E-17E, wherein thetunable detection system detects, measures, or quantifies the level ofbinding and/or amount of one or more targets present in a sample fromthe generated signal by one or more of the following: flow cytometry,microscopy, imaging, high content screening (HCS), multiplex bead array,microarray, antibody array, cellular array, immunohistochemistry (IHC),immunocytochemistry (ICC), in situ hybridization (ISH), enzymeimmuno-assay (EIA), enzyme linked immuno-assay (ELISA), ELISpot, orblotting method

Example 19E

The tunable detection system of any one of Examples 10E-18E, wherein thetunable detection system minimizes signal spillover by varying one ormore of the following: the identity of the first signal generatingmoiety, the number of the first signal generating moieties on the atleast first detectable component, the identity of the second signalgenerating moiety, the number of the second signal generating moietieson the at least second detectable component.

Example 20E

The tunable detection system of any one of Examples 1E-19E, wherein thesample comprises a plurality of targets.

Example 21E

The tunable detection system of any one of Examples 1E-20E, wherein thetunable detection system comprises one or more of the following: i) theplurality of molecular probes comprises the plurality of bindingmoieties independently conjugated to a plurality of oligonucleotidesequences, comprising: 1) at least a first molecular probe comprisingthe first binding moiety conjugated to a first oligonucleotide sequence;and 2) at least a second molecular probe comprising the second bindingmoiety conjugated to a second oligonucleotide sequence; and ii) theplurality of detectable components comprising the range of first signalgenerating moieties independently conjugated to the universaloligonucleotide sequence, comprising: 1) at least a first detectablecomponent comprising the range of first signal generating moietiesconjugated to the universal oligonucleotide sequence, wherein the rangeof first signal generating moieties generates a range of signalintensities; and 2) at least a second detectable component comprisingthe range of second signal generating moieties conjugated to theuniversal oligonucleotide sequence, wherein the range of second signalgenerating moieties generates a range of signal intensities; and iii) aplurality of universal adapters, comprising a plurality ofoligonucleotide sequence segments complementary to the plurality ofoligonucleotide sequence of the plurality of molecular probesindependently paired with a second oligonucleotide sequence segmentcomplementary to the universal oligonucleotide sequence.

Example 22E

The tunable detection system of any one of Examples 1E-21E, wherein theplurality of targets comprises substantially similar or substantiallydifferent targets.

Example 23E

The tunable detection system of any one of Examples 1E-22E, wherein atleast a first target of the plurality of targets is different among theplurality of targets.

Example 24E

The tunable detection system of any one of Examples 1E-23E, wherein thetunable detection system comprises an automated system or roboticsystem.

Example 25E

The tunable detection system of any one of Examples 1E-24E, wherein thetunable detection system further comprises removing the hybridizeddetectable component or plurality of detectable components from thebound target or plurality of targets, respectively, wherein said removalis by a washing or stripping process.

Example 26E

The tunable detection system of Example 25E, wherein the removalcomprises de-hybridizing the detectable component or the plurality ofdetectable components, respectively.

Example 27E

The tunable detection system of Examples 25E or 26E, wherein the tunabledetection system further comprises re-probing with a second detectablecomponent or second plurality of detectable components, respectively,wherein said second detectable component comprises at least one secondsignal generating moieties conjugated to a second oligonucleotidesequence or a complementary second oligonucleotide sequence, or saidsecond plurality of detectable components are prepared by independentlypairing, via conjugation, a second plurality of signal generatingmoieties and a second plurality of second oligonucleotide sequences or asecond plurality of complementary second oligonucleotide sequences.

Example 28E

The tunable detection system of any one of Examples 1E-27E, wherein therange of different signals generated from the signal generating moietiesor plurality of signal generating moieties comprises between 2-20.

Example 29E

The tunable detection system of any one of Examples 1E-28E, wherein therange of different signals generated from the signal generating moietiesor plurality of signal generating moieties comprises between 2-10.

Example 30E

The tunable detection system of any one of Examples 1E-29E, wherein theintensity of the signal generated can be tuned over a range 1.25 to 2×.

Example 31E

The tunable detection system of any one of Examples 1E-30E, wherein theintensity of the signal generated can be tuned over a range 1.5 to 3×.

Example 32E

The tunable detection system of any one of Examples 1E-31E, wherein theintensity of the signal generated can be tuned over a range 2 to 4×.

Example 33E

The tunable detection system of any one of Examples 1E-32E, wherein theintensity of the signal generated can be tuned over a range 1.25 to1.75×.

Example 34E

The tunable detection system of any one of Examples 1E-33E, wherein theintensity of the signal generated can be tuned over a range 2 to 6×.

Example 35E

The tunable detection system of any one of Examples 1E-34E, wherein theintensity of the signal generated can be tuned over a range 3 to 5×.

Example 36E

The tunable detection system of any one of Examples 1E-35E, wherein theintensity of the signal generated can be tuned over a range 2 to 10×.

Example 37E

The tunable detection system of any one of Examples 1E-36E, wherein thebinding moiety comprises an antibody, a monoclonal antibody, apolyclonal antibody, an enzyme, a protein, a peptide, a carbohydrate, anuclear receptor, a small molecule, an aptamer, a chelator, orcombinations or derivatives thereof.

Example 1F

A manufacturing system, comprising: i) a first series, comprising aplurality of molecular probes, said first series prepared byindependently pairing, via conjugation, a plurality of firstoligonucleotide sequences to a plurality of binding moieties; and ii) asecond series, comprising a plurality of detectable components, saidsecond series prepared by independently pairing, via conjugation, aplurality of second oligonucleotide sequences to a plurality of signalgenerating moieties or to a plurality of scaffolds having one or more ofthe plurality of signal generating moieties, wherein the plurality ofsecond oligonucleotide sequences are complementary to the plurality offirst oligonucleotide sequences; wherein the manufacturing system ischaracterized by one or more of the following: a) the first series andthe second series are made available for one or more users to combinethe first series and the second series to produce one or more hybridizedmolecular probes; b) at least a portion of preassembled combinations ofthe first series and the second series are produced and made availablefor one or more users; c) the first series and the second series aremade available for one or more users to combine the first series, thesecond series, and a sample potentially having one or more targets, toproduce one or more hybridized target-bound molecular probes; d) thetime in which to produce the possible combinations of said first seriesand said second series is less than that of conventional preparations;and e) the time in which to hybridize and detect of the target-boundhybrids formed from said first series and said second series is lessthan conventional conjugation and detection.

Example 2F

The manufacturing system of Example 1F, wherein the manufacturing systemis further characterized by one or more of the following: i) the firstseries and/or second series is provided to one or more end users as acustomized matrix or semi-matrix of the first series and the secondseries as independently selected and paired by said one or more endusers, wherein the customized matrix or semi-matrix comprises an assayuseful amount of said first series and said second series which arecapable of producing a plurality of hybridized molecularprobe-detectable components; ii) the manufacturing system reduces tomanageable proportions the number of catalog products a vendor oflabeled molecular probes must manufacture, stock, market, anddistribute; and iii) at least 90% of the possible hybridizedcombinations of said first series and second series can be produced in10 hours or less.

Example 3F

A manufacturing system, comprising: i) a first series, comprising aplurality of molecular probes, said first series prepared byindependently pairing, via conjugation, a plurality of firstoligonucleotide sequences to a plurality of binding moieties; ii) asecond series, comprising a plurality of detectable components, saidsecond series prepared by independently pairing, via conjugation, aplurality of second oligonucleotide sequences to a plurality of signalgenerating moieties or to a plurality of scaffolds having one or more ofthe plurality of signal generating moieties; and iii) a third series,comprising a plurality of universal adapters comprising a plurality ofcomplementary first oligonucleotide sequence segments independentlypaired with a plurality of complementary second oligonucleotide sequencesegments; wherein the manufacturing system is characterized by one ormore of the following: a) the first series, the second series, and thethird series, are made available for one or more users to combine thefirst series, the second series, and the third series, to produce one ormore hybridized molecular probes; b) at least a portion of preassembledcombinations of the first series, the second series, and the thirdseries, are produced and made available for one or more users; c) thefirst series, the second series, and the third series, are madeavailable for one or more users to combine the first series, the secondseries, the third series, and a sample potentially having one or moretargets, to produce one or more hybridized target-bound molecularprobes; d) the time in which to produce the possible combinations ofsaid first series, said second series, and said third series, is lessthan that of conventional preparations; and e) the time in which tohybridize and detect of the target-bound hybrids formed from said firstseries, said second series, and said third series, is less thanconventional conjugation and detection.

Example 4F

The manufacturing system of Example 3F, wherein the manufacturing systemis further characterized by one or more of the following: i) the firstseries, second series, and/or third series is provided to one or moreend users as a customized matrix or semi-matrix of the first series, thesecond series, and the third series, as independently selected andpaired by said one or more end users, wherein the customized matrix orsemi-matrix comprises an assay useful amount of said first series, saidsecond series, and said third series which are capable of producing aplurality of hybridized molecular probe-detectable components; ii) themanufacturing system reduces to manageable proportions the number ofcatalog products a vendor of labeled molecular probes must manufacture,stock, market, and distribute; and iii) at least 90% of the possiblehybridized combinations of said first series, second series, and thirdseries can be produced in 10 hours or less.

Example 5F

The manufacturing system of any one of Examples 1F-4F, wherein the firstseries comprises a plurality of between 2-50 different molecular probes.

Example 6F

The manufacturing system of any one of Examples 1F-5F, wherein the firstseries comprises a plurality of between 2-25 different molecular probes.

Example 7F

The manufacturing system of any one of Examples 1F-6F, wherein the firstseries comprises: i) a plurality of different first oligonucleotidesequences; or ii) a plurality of identical first oligonucleotidesequences.

Example 8F

The manufacturing system of any one of Examples 1F-7F, wherein thesecond series comprises a plurality of between 2-50 different detectablecomponents.

Example 9F

The manufacturing system of any one of Examples 1F-8F, wherein thesecond series comprises a plurality of between 2-25 different detectablecomponents.

Example 10F

The manufacturing system of any one of Examples 1F-9F, wherein thesecond series comprises a plurality of scaffolds independently paired,via conjugation, with the plurality of second oligonucleotide sequences,wherein said plurality of scaffolds comprise one or more signalgenerating moieties.

Example 11F

The manufacturing system of any one of Examples 1F-10F, wherein thesecond series comprises: i) a plurality of different secondoligonucleotide sequences; or ii) a plurality of identical secondoligonucleotide sequences.

Example 12F

The manufacturing system of any one of Examples 3F-11F, wherein thethird series comprises a plurality of between 2-50 different universaladapters.

Example 13F

The manufacturing system of any one of Examples 3F-12F, wherein thethird series comprises a plurality of between 2-25 different universaladapters.

Example 14F

The manufacturing system of any one of Examples 3F-13F, wherein thethird series comprises: i) a plurality of different complementary firstoligonucleotide sequence segments; and. ii) a plurality of identicalcomplementary second oligonucleotide sequence segments.

Example 15F

The manufacturing system of any one of Examples 3F-14F, wherein thethird series comprises: i) a plurality of identical complementary firstoligonucleotide sequence segments; and. ii) a plurality of differentcomplementary second oligonucleotide sequence segments.

Example 16F

The manufacturing system of any one of Examples 1F-15F, wherein themanufacturing system produces at least one or more of the following: i)a customized first series; and ii) a customized second series; whereinthe manufacturing system reduces to manageable proportions the number ofcatalog products a vendor of labeled molecular probes and/or detectablecomponents to manufacture, stock, market, and/or distribute.

Example 17F

The manufacturing system of Example 16F, wherein the manufacturingsystem further produces a customized third series.

Example 18F

The manufacturing system of any one of Examples 1F-17F, wherein themanufacturing system enables a manufacturer to provide at least one ormore of the following: i) a plurality of customizable molecular probes,comprising a plurality of first oligonucleotide sequences conjugated toa plurality of binding moieties; and ii) a plurality of customizabledetectable components, comprising a plurality of second oligonucleotidesequences conjugated to a plurality of signal generating moieties.

Example 19F

The manufacturing system of Example 18F, wherein the manufacturingsystem further enables a manufacturer to provide a plurality ofcustomizable universal adapters, comprising a plurality of firstcomplementary oligonucleotide sequence segments independently pairedwith a plurality of second complementary oligonucleotide sequencesegments.

Example 20F

The manufacturing system of any one of Examples 1F-19F, wherein themanufacturing system enables either the manufacturer or the end user toproduce the plurality of customizable molecular probes and/or theplurality of customizable detectable components in a time at least 10%less, 20%, 30%, 40%, 50%, 75%, 100%, 200%, 300%, or 500% less than asrequired by conventional conjugations to prepare the directly labeledbinding moieties.

Example 21F

The manufacturing system of Example 20F, wherein the manufacturingsystem further enables either the manufacturer or the end user toproduce the plurality of customizable molecular probes, the plurality ofcustomizable detectable components, and/or the plurality of customizableuniversal adapters in a time at least 10% less, 20%, 30%, 40%, 50%, 75%,100%, 200%, 300%, or 500% less than as required by conventionalconjugations to prepare the directly labeled binding moieties.

Example 22F

The manufacturing system of any one of Examples 1F-21F, wherein themanufacturing system enables either the manufacturer or the end user tohybridize the plurality of customizable molecular probes and theplurality of customizable detectable components to form a plurality ofhybridized combinations in a time at least 10% less, 20%, 30%, 40%, 50%,75%, 100%, 200%, 300%, or 500% less than as required by conventionalconjugations to prepare the directly labeled binding moieties

Example 23F

The manufacturing system of Example 22F, wherein the manufacturingsystem further enables either the manufacturer or the end user tohybridize the plurality of customizable molecular probes, the pluralityof customizable detectable components, and the plurality of customizableuniversal adapters to form a plurality of hybridized combinations in atime at least 10% less, 20%, 30%, 40%, 50%, 75%, 100%, 200%, 300%, or500% less than as required by conventional conjugations to prepare thedirectly labeled binding moieties.

Example 24F

The manufacturing system of any one of Examples 1F-23F, wherein, priorto contacting a sample comprising one or more targets, the manufacturingsystem enables the end user to hybridize the plurality of customizablemolecular probes and the plurality of customizable detectablecomponents, to form a plurality of hybridized combinations a time atleast 10% less, 20%, 30%, 40%, 50%, 75%, 100%, 200%, 300%, or 500% lessthan as required by conventional conjugations to prepare the directlylabeled binding moieties.

Example 25F

The manufacturing system of Example 24F, wherein, prior to contacting asample comprising one or more targets, the manufacturing system furtherenables the end user to hybridize the plurality of customizablemolecular probes, the plurality of customizable detectable components,and the plurality of customizable universal adapters to form a pluralityof hybridized combinations in a time at least 10% less, 20%, 30%, 40%,50%, 75%, 100%, 200%, 300%, or 500% less than as required byconventional conjugations to prepare the directly labeled bindingmoieties.

Example 26F

The manufacturing system of any one of Examples 1F-23F, wherein themanufacturing system enables the end user, after contacting a samplecomprising one or more targets with either: i) the plurality ofcustomizable molecular probes; or ii) the plurality of customizabledetectable components; to hybridize the plurality of customizablemolecular probes, either bound or non-bound to at least one of the oneor more targets, and the plurality of customizable detectable componentsin the presence of the sample to form a plurality of hybridizedcombinations and/or a plurality of target-bound hybridized combinationsin a time at least 10% less, 20%, 30%, 40%, 50%, 75%, 100%, 200%, 300%,or 500% less than as required by conventional conjugations to preparethe directly labeled binding moieties.

Example 27F

The manufacturing system of Example 26F, wherein the manufacturingsystem further enables the end user, after contacting a samplecomprising one or more targets with either: i) the plurality ofcustomizable molecular probes; ii) the plurality of customizabledetectable components; or iii) the plurality of customizable universaladapters; to hybridize the plurality of customizable molecular probes,either bound or non-bound to at least one of the one or more targets,and the plurality of customizable detectable components and theplurality of customizable universal adapters in the presence of thesample to form a plurality of hybridized combinations and/or a pluralityof target-bound hybridized combinations a time at least 10% less, 20%,30%, 40%, 50%, 75%, 100%, 200%, 300%, or 500% less than as required byconventional conjugations to prepare the directly labeled bindingmoieties.

Example 28F

The manufacturing system of any one of Examples 1F-27F, wherein themanufacturing system enables the end user to choose the mode of additionof the first series and the second series to the sample comprising oneor more targets.

Example 29F

The manufacturing system of any one of Examples 3F-28F, wherein themanufacturing system enables the end user to choose the mode of additionof the first series, the second series, and the third series to thesample comprising one or more targets.

Example 30F

The manufacturing system of any one of Examples 1F-29F, wherein themanufacturing system enables the end user to pre-assemble, viahybridization, prior to contacting the sample, in a time at least 10%less, 20%, 30%, 40%, 50%, 75%, 100%, 200%, 300%, or 500% less than asrequired by conventional conjugations to prepare the directly labeledbinding moieties.

Example 31F

The manufacturing system of any one of Examples 1F-30F, wherein themanufacturing system enables either the manufacturer or the end user toproduce a complete or semi-complete matrix of combination of said firstseries and said second series, in a time at least 10% less, 20%, 30%,40%, 50%, 75%, 100%, 200%, 300%, or 500% less than as required byconventional conjugations to prepare the directly labeled bindingmoieties.

Example 32F

The manufacturing system of Example 31F, wherein the manufacturingsystem further enables either the manufacturer or the end user toproduce a complete or semi-complete matrix of combination of said firstseries, said second series, and said third series, in a time at least10% less, 20%, 30%, 40%, 50%, 75%, 100%, 200%, 300%, or 500% less thanas required by conventional conjugations to prepare the directly labeledbinding moieties

Example 33F

The manufacturing system of any one of Examples 1F-32F, wherein themanufacturing system enables a vendor or manufacturer to reduce tomanageable proportions the number of products the vendor or themanufacturer of molecular probes, detectable components, and/oruniversal detectors, must produce, stock, market, and/or distribute,than vendors or manufacturers using conventional conjugations systems toprepare the directly labeled binding moieties.

Example 34F

The manufacturing system of any one of Examples 1F-33F, wherein themanufacturing system enables a vendor or manufacturer to offer a greatervariety of molecular probes, detectable components, and/or universaldetectors, than vendors or manufacturers using conventional conjugationssystems to prepare the directly labeled binding moieties.

Example 35F

The manufacturing system of any one of Examples 3F-34F, wherein themanufacturing system enables a vendor or manufacturer to prepare alarger number and/or a more diverse set of molecular probes, detectablecomponents, and/or universal detectors, than vendors or manufacturersusing conventional conjugations systems to prepare the directly labeledbinding moieties.

Example 36F

The manufacturing system of any one of Examples 1F-35F, wherein themanufacturing system enables a vendor or manufacturer to produce acustomizable catalog of molecular probes, detectable components, anduniversal detectors, as compared to vendors or manufacturers usingconventional conjugations systems to prepare the directly labeledbinding moieties.

Example 37F

The manufacturing system of any one of Examples 1F-36F, wherein themanufacturing system enables a vendor or manufacturer to rapidly,reproducibly, and on demand, produce molecular probes having, onaverage, between about 1-4 oligonucleotide sequences conjugated onto abinding moiety.

Example 38F

The manufacturing system of any one of Examples 1F-37F, wherein themanufacturing system enables a vendor or manufacturer to rapidly,reproducibly, and on demand, produce detectable components having apredetermined number of signal generating moieties conjugated to thecomplementary second oligonucleotide sequences or the secondoligonucleotide sequences.

Example 39F

The manufacturing system of any one of Examples 1F-38F, wherein themanufacturing system enables a vendor or manufacturer to rapidly,reproducibly, and on demand, produce detectable components having apredetermined number of scaffolds conjugated to the complementary secondoligonucleotide sequences or the second oligonucleotide sequences,wherein the scaffolds comprise a plurality of signal generatingmoieties.

Example 40F

The manufacturing system of any one of Examples 1F-39F, wherein themanufacturing system enables a vendor or manufacturer to rapidly,reproducibly, and on demand, produce universal adapters having either:i) a complementary first oligonucleotide sequence segment independentlypaired with a plurality of different complementary secondoligonucleotide sequence segments; or ii) a plurality of differentcomplementary first oligonucleotide sequence segments independentlypaired with a complementary second oligonucleotide sequence segment.

Example 41F

The manufacturing system of any one of Examples 1F-40F, wherein theconjugation of least one of the following: i) between the firstoligonucleotide sequence and the binding moiety of the first series; ii)between the second complementary oligonucleotide sequence and the signalgenerating moiety or the scaffold comprising the signal generatingmoieties of the second series; and ii) between the secondoligonucleotide sequence and the signal generating moiety or thescaffold comprising the signal generating moieties of the second serieswhen the third series is employed; comprises one or more covalent bondlinkages, comprising a hydrazone, oxime, triazine, or other covalentbond, wherein the formation of the conjugates are at least 90%efficient.

Example 42F

The manufacturing system of any one of Examples 1F-41F, whereinmanufacturing system enables the end user to prepare and utilize anassay comprising: i) a singleplex or multiplex assay; and ii) the assaydetects, measures, or quantifies the level of binding and/or amount ofthe target present in the sample with one or more of the following: flowcytometry, immunomagnetic cellular depletion, immunomagnetic cellcapture, array, bead array, multiplex bead array, microarray, antibodyarray, cellular array, chemiluminescence, infrared, microscopy, imaging,high content screening (HCS), mass cytometry, lateral flow immunoassay,immunodetection, immunoturbidity, latex agglutination, gold particleagglutination, visual inspection, a change in light transmittancethrough said sample, increased light transmittance through said sample,immunohistochemistry (IHC), immunocytochemistry (ICC), in situhybridization (ISH), enzyme immuno-assay (EIA), enzyme linkedimmuno-assay (ELISA), ELISpot, a blotting method, a Western blot, aSouthern blot, a Southwestern blot, labeling inside an electrophoresissystem, labeling on a surface, labeling on an array, PCR amplification,elongation followed by PCR amplification, immunoprecipitation,co-immunoprecipitation, chromatin immunoprecipitation, pretargetingimaging, therapeutic agent, or combinations thereof.

Example 43F

The manufacturing system of any one of Examples 1F-42F, whereinmanufacturing system enables the end user to prepare and utilize anassay comprising: i) a singleplex or multiplex assay; and ii) the assaydetects, measures, or quantifies the level of binding and/or amount ofthe target present in the sample with one or more of the following: flowcytometry, microscopy, imaging, high content screening (HCS), multiplexbead array, microarray, antibody array, cellular array,immunohistochemistry (IHC), immunocytochemistry (ICC), in situhybridization (ISH), enzyme immuno-assay (EIA), enzyme linkedimmuno-assay (ELISA), ELISpot, or a blotting method.

Example 44F

The manufacturing system of any one of Examples 1F-43F, whereinmanufacturing system enables the end user to prepare and utilize anassay comprising: i) a singleplex or multiplex assay; and ii) the assaydetects, measures, or quantifies the level of binding and/or amount ofthe target present in the sample with one or more of the following: flowcytometry, mass cytometry, lateral flow immunoassay,immunohistochemistry (IHC), immunocytochemistry (ICC),immunoprecipitation, pretargeting imaging, therapeutic agent, orcombinations thereof.

Example 45F

The manufacturing system of any one of Examples 1F-44F, whereinmanufacturing system enables the end user to analyze a samplecharacterized as at least one or more of the following: i) a complexsample; and ii) a homogeneous or a heterogeneous mixture; wherein saidsample comprises at least one or more of the following: a) one or moreanalytes having substantially the same or substantially differentbinding specificities; b) one or more of the following biologiccomponents, comprising: cells, membranes, biological molecules,metabolites, or disease biomarkers; and c) a biological fluid or afluidized biological tissue.

Example 46F

The manufacturing system of any one of Examples 1F-45F, wherein at leastone of the molecular probes produced by the manufacturing systemcomprises one or more of the following properties: i) a molecular weightof between about 15,000 Daltons to about 450,000 Daltons; ii) asolubility that is substantially the same as that of the unconjugatedbinding moiety; iii) a solubility that minimizes non-specific binding tothe target; iv) the first oligonucleotide sequence of the molecularprobe does not adversely affect the solubility of the binding moiety; v)interacts and binds to the target via interactions other thanexclusively electrostatic; vi) a unique, distinguishable, and/orspecifically designed oligonucleotide sequence; and vii) the firstoligonucleotide sequence of the molecular probe is uniquely andspecifically designed to hybridize to the second oligonucleotidesequence of the detectable component.

Example 47F

The manufacturing system of any one of Examples 1F-46F, wherein themanufacturing system wherein the prepared molecular probes and prepareddetectable components have at least 90% purity.

Example 48F

The manufacturing system of any one of Examples 3F-47F, wherein themanufacturing system wherein the prepared universal adapters have atleast 90% purity.

Example 49F

The manufacturing system of any one of Examples 1F-48F, wherein thepreparation further comprises an isolation step utilizing an immobilizedbinder, chromatography, affinity chromatography, size exclusionchromatography, HPLC, reverse-phase chromatography, electrophoresis,capillary electrophoresis, polyacrylamide gel electrophoresis, agarosegel electrophoresis, free flow electrophoresis, differentialcentrifugation, thin layer chromatography, immunoprecipitation,hybridization, solvent extraction, dialysis, filtration, diafiltration,tangential flow filtration, ion exchange chromatography, hydrophobicinteraction chromatography, or combinations thereof.

Example 50F

The manufacturing system of Example 49F, wherein the isolation steputilizes an immobilized binder, affinity chromatography, size exclusionchromatography, electrophoresis, differential centrifugation,immunoprecipitation, hybridization, solvent extraction, dialysis,filtration, diafiltration, ion exchange chromatography, hydrophobicinteraction chromatography, or combinations thereof.

Example 51F

The manufacturing system of Example 49F, wherein the isolation steputilizes an immobilized binder, affinity chromatography, size exclusionchromatography, differential centrifugation, dialysis, filtration,hydrophobic interaction chromatography, or combinations thereof.

Example 52F

The manufacturing system of any one of Examples 1F-51F, wherein thebinding moiety comprises an antibody, a monoclonal antibody, apolycolonal antibody, an enzyme, a protein, a peptide, a carbohydrate, anuclear receptor, a small molecule, an aptamer, a chelator, orcombinations or derivatives thereof.

Example 53F

The manufacturing system of any one of Examples 1F-52F, wherein thescaffold comprises a dendrimer, a polysaccharide, a dextran, a protein,a peptide, a further oligonucleotide sequence, a portion of the secondoligonucleotide sequence that is not complementary to the firstoligonucleotide sequence of the molecular probe, a polymer, ahydrophilic polymer, a bead, a nanoparticle, or combinations orderivatives thereof.

Example 54F

The manufacturing system of any one of Examples 1F-53F, wherein thesignal generating moiety or the one or more signal generating moietiesof the detectable component or the hybridized detectable component,comprises one or more of the following: a directly detectable signalgenerating moiety, an indirectly detectable signal generating moiety, afluorescent dye, a fluorophore, a fluorochrome, a chromophore, abiofluorescent protein, a luminescent species, a chemiluminescentcompound, a electrochemiluminescent label, a bioluminescent label, aphosphorescent species, a fluorophore labeled DNA dendrimer, QuantumDot, a tandem dye, a FRET dye, a heavy atom, a spin label, a radioactiveisotope, a nanoparticle, a light scattering nanoparticle or microsphere,a diffracting particle, a polymer, a polymer particle, a bead, a solidsurface, a Raman particle, a metal particle, a stable isotope, a heavymetal chelate, a magnetic particle, an RFID tag, a microbarcodeparticle, an enzyme, an enzyme substrate, a molecule specificallyrecognized by another substance carrying a label or reacts with asubstance carrying a label, an antibody, an antibody fragment, anantigen, a nucleic acid, a nucleic acid analog, oligonucleotide,oligonucleotide analog, complementary oligonucleotide, complementaryoligonucleotide analog, a ligand, a protein, a peptide ligand, a proteinsubstrate, a receptor; a substrate, a secondary reporter, a hapten, orcombinations or derivatives thereof.

Example 55F

The manufacturing system of any one of Examples 1F-54F, wherein the oneor more signal generating moieties provides an enhanced signal thatminimizes detection errors from background noise, relative toconventionally labeled binding moieties.

Example 56F

The manufacturing system of any one of Examples 1F-66F, wherein themolecular probe, the detectable component, and/or universal adapterfurther comprises a spacer group, comprising a polymerized ethyleneoxide, a PEG, a PEO, a protein, a peptide, a DNA, an RNA, anoligonucleotide sequence, or a dextran.

Example 57F

The manufacturing system of any one of Examples 1F-56F, wherein thebinding moiety, the scaffold, the first oligonucleotide sequence, and/orthe second oligonucleotide sequence comprise HyNic or 4-FB.

Example 58F

The manufacturing system of any one of Examples 1F-57F, wherein thedetectable component comprises a unique, distinguishable, and/orspecifically designed second oligonucleotide sequence.

Example 59F

The manufacturing system of any one of Examples 1F-58F, wherein thefirst oligonucleotide sequence, second oligonucleotide sequence, and/oroligonucleotide sequence segment comprising an oligonucleotide sequenceconjugated at the 3′-position, an oligonucleotide sequence conjugated atthe 5′-position, linear oligonucleotide sequences, branchedoligonucleotide sequences, LNAs, PNAs, oligonucleotide sequencesoptionally covalently attached to other moieties, or combinations orderivatives thereof.

Example 60F

The manufacturing system of any one of Examples 1F-58F, wherein themanufacturing system enables the end user to analyze a sample comprisingone or more targets.

Example 61F

The manufacturing system of Example 59F, wherein at least one target ofthe one or more targets is a biological target.

Example 62F

The manufacturing system of Example 61F, wherein the biological targetcomprises an antigen, a pathogen, a protein, a peptide, an epitope, acarbohydrate-containing molecule, a small molecule, or combinations orderivatives thereof.

Example 63F

The manufacturing system of any one of Examples 1F-62F, wherein themanufacturing system comprises an automated system or robotic system toprepare at least one of the following: the first series, the secondseries, and the third series.

Example 64F

The manufacturing system of any one of Examples 1F-62F, wherein themanufacturing system enables an end user to utilize an automated systemor robotic system to prepare hybridized combinations of at least one ofthe following: i) the first series and the second series; ii) the firstseries and the third series; iii) the second series and the thirdseries; and iv) the first series, the second series, and the thirdseries.

Example 65F

The manufacturing system of Example 64F, wherein the hybridizedcombinations are prepared prior to contacting the sample or preparedwhile in contact with the sample.

Example 66F

The manufacturing system of any one of Examples 1F-65F, wherein themanufacturing system further enables the end user to remove thehybridized detectable component or plurality of detectable componentsfrom the bound target or plurality of targets, respectively, whereinsaid removal is by a washing or stripping process.

Example 67F

The manufacturing system of Example 66F, wherein the removal comprisesde-hybridizing the detectable component or the plurality of detectablecomponents, respectively.

Example 68F

The manufacturing system of Examples 66F or 67F, wherein themanufacturing system further enables re-probing with a second detectablecomponent or second plurality of detectable components, respectively,wherein said second detectable component comprises at least one secondsignal generating moieties conjugated to a second oligonucleotidesequence or a complementary second oligonucleotide sequence, or saidsecond plurality of detectable components are prepared by independentlypairing, via conjugation, a second plurality of signal generatingmoieties and a second plurality of second oligonucleotide sequences or asecond plurality of complementary second oligonucleotide sequences.

Example 69F

A manufacturing method, comprising: i) a first series, comprising aplurality of molecular probes, said first series prepared byindependently pairing, via conjugation, a plurality of firstoligonucleotide sequences to a plurality of binding moieties; and ii) asecond series, comprising a plurality of detectable components, saidsecond series prepared by independently pairing, via conjugation, aplurality of second oligonucleotide sequences to a plurality of signalgenerating moieties or to a plurality of scaffolds having one or more ofthe plurality of signal generating moieties, wherein the plurality ofsecond oligonucleotide sequences are complementary to the plurality offirst oligonucleotide sequences; wherein the manufacturing system ischaracterized by one or more of the following: a) the first series andthe second series are made available for one or more users to combinethe first series and the second series to produce one or more hybridizedmolecular probes; b) at least a portion of preassembled combinations ofthe first series and the second series are produced and made availablefor one or more users; c) the first series and the second series aremade available for one or more users to combine the first series, thesecond series, and a sample potentially having one or more targets, toproduce one or more hybridized target-bound molecular probes; d) thetime in which to produce the possible combinations of said first seriesand said second series is less than that of conventional preparations;and e) the time in which to hybridize and detect of the target-boundhybrids formed from said first series and said second series is lessthan conventional conjugation and detection.

Example 70F

The manufacturing method of Example 69F, wherein the method furthercomprises providing to one or more end users a customized matrix orsemi-matrix of the first series and the second series as independentlyselected and paired by said one or more end users, wherein thecustomized matrix or semi-matrix comprises an assay useful amount ofsaid first series and said second series which are capable of producinga plurality of hybridized molecular probe-detectable components;wherein: a) the manufacturing method reduces to manageable proportionsthe number of catalog products a vendor of labeled molecular probes mustmanufacture, stock, market, and distribute; and b) at least 90% of thepossible hybridized combinations of said first series and second seriescan be produced in 10 hours or less.

Example 71F

A manufacturing method, comprising: i) preparing a first series,comprising a plurality of molecular probes, said first series preparedby independently pairing, via conjugation, a plurality of firstoligonucleotide sequences to a plurality of binding moieties; ii)preparing a second series, comprising a plurality of detectablecomponents, said second series prepared by independently pairing, viaconjugation, a plurality of second oligonucleotide sequences to aplurality of signal generating moieties or to a plurality of scaffoldshaving one or more of the plurality of signal generating moieties; andiii) preparing a third series, comprising a plurality of universaladapters comprising a plurality of complementary first oligonucleotidesequence segments independently paired with a plurality of complementarysecond oligonucleotide sequence segments; wherein the method ischaracterized by one or more of the following: a) the prepared firstseries, the prepared second series, and the prepared third series, aremade available for one or more users to combine the prepared firstseries, the prepared second series, and the prepared third series, toproduce one or more hybridized molecular probes; b) at least a portionof preassembled combinations of the prepared first series, the preparedsecond series, and the prepared third series, are produced and madeavailable for one or more users; c) the prepared first series, theprepared second series, and the prepared third series, are madeavailable for one or more users to combine the prepared first series,the prepared second series, the prepared third series, and a samplepotentially having one or more targets, to produce one or morehybridized target-bound molecular probes; d) the time in which toproduce the possible combinations of said prepared first series, saidprepared second series, and said prepared third series, is less thanthat of conventional preparations; and e) the time in which to hybridizeand detect of the target-bound hybrids formed from said prepared firstseries, said prepared second series, and said prepared third series, isless than conventional conjugation and detection.

Example 72F

The manufacturing method of Example 71F, wherein the method furthercomprises providing to one or more end users a customized matrix orsemi-matrix of the first series, the second series, and the thirdseries, as independently selected and paired by said one or more endusers, wherein the customized matrix or semi-matrix comprises an assayuseful amount of said first series, said second series, and said thirdseries which are capable of producing a plurality of hybridizedmolecular probe-detectable components; wherein: a) the manufacturingmethod reduces to manageable proportions the number of catalog productsa vendor of labeled molecular probes must manufacture, stock, market,and distribute; and b) at least 90% of the possible hybridizedcombinations of said first series, second series, and third series canbe produced in 10 hours or less.

Example 73F

A method of offering detectable molecular probes, comprising: i)offering to one or more users a first series, comprising a plurality ofmolecular probes, said first series prepared by independently pairing,via conjugation, a plurality of first oligonucleotide sequences to aplurality of binding moieties; ii) offering to one or more users asecond series, comprising a plurality of detectable components, saidsecond series prepared by independently pairing, via conjugation, aplurality of second oligonucleotide sequences to a plurality of signalgenerating moieties or to a plurality of scaffolds having one or more ofthe plurality of signal generating moieties, wherein the plurality ofsecond oligonucleotide sequences are complementary to the plurality offirst oligonucleotide sequences; and iii) providing the first series andthe second series to the one or more users, to combine the first series,the second series, and a sample having one or more targets, at anappropriate time, and in an appropriate amount, to produce one or morehybridized target-bound molecular probes; wherein the method ischaracterized by one or more of the following: a) the first series andthe second series are made available for one or more users to combinethe first series and the second series to produce one or more hybridizedmolecular probes; b) at least a portion of preassembled combinations ofthe first series and the second series are produced and made availablefor one or more users; c) the first series and the second series aremade available for one or more users to combine the first series, thesecond series, and a sample potentially having one or more targets, toproduce one or more hybridized target-bound molecular probes; d) thetime in which to produce the possible combinations of said first seriesand said second series is less than that of conventional preparations;and e) the time in which to hybridize and detect of the target-boundhybrids formed from said first series and said second series is lessthan conventional conjugation and detection.

Example 74F

The method of Example 73F, wherein: i) the first series and/or secondseries is provided to the one or more end users as a customized matrixor semi-matrix of the first series and the second series, independentlyselected and paired by said one or more end users, wherein thecustomized matrix or semi-matrix comprises an assay useful amount ofsaid first series and said second series which are capable of producinga plurality of hybridized molecular probe-detectable components; ii) themethod reduces to manageable proportions the number of catalog productsa vendor of labeled molecular probes must manufacture, stock, market,and distribute; and iii) at least 90% of the possible hybridizedcombinations of said first series and second series can be produced in10 hours or less.

Example 75F

A method of offering detectable molecular probes, comprising: i)offering to one or more users a first series, comprising a plurality ofmolecular probes, said first series prepared by independently pairing,via conjugation, a plurality of first oligonucleotide sequences to aplurality of binding moieties; ii) offering to one or more users asecond series, comprising a plurality of detectable components, saidsecond series prepared by independently pairing, via conjugation, aplurality of second oligonucleotide sequences to a plurality of signalgenerating moieties or to a plurality of scaffolds having one or more ofthe plurality of signal generating moieties; and iii) offering to one ormore users a third series, comprising a plurality of universal adapterscomprising a plurality of complementary first oligonucleotide sequencesegments independently paired with a plurality of complementary secondoligonucleotide sequence segments; iii) providing the first series, thesecond series, and the third series to the one or more users, to combinethe first series, the second series, the third series and a samplehaving one or more targets, at an appropriate time, and in anappropriate amount, to produce one or more hybridized target-boundmolecular probes; wherein the method is characterized by one or more ofthe following: a) the first series and the second series are madeavailable for one or more users to combine the first series and thesecond series to produce one or more hybridized molecular probes; b) atleast a portion of preassembled combinations of the first series and thesecond series are produced and made available for one or more users; c)the first series and the second series are made available for one ormore users to combine the first series, the second series, and a samplepotentially having one or more targets, to produce one or morehybridized target-bound molecular probes; d) the time in which toproduce the possible combinations of said first series and said secondseries is less than that of conventional preparations; and e) the timein which to hybridize and detect of the target-bound hybrids formed fromsaid first series and said second series is less than conventionalconjugation and detection.

Example 76F

The method of Example 75F, wherein: i) the first series, the secondseries, and/or the third series is provided to the one or more end usersas a customized matrix or semi-matrix of the first series, the secondseries, and the third series, as independently selected and paired bysaid one or more end users, wherein the customized matrix or semi-matrixcomprises an assay useful amount of said first series, said secondseries, and said third series which are capable of producing a pluralityof hybridized molecular probe-detectable components; ii) the methodreduces to manageable proportions the number of catalog products avendor of labeled molecular probes must manufacture, stock, market, anddistribute; and iii) at least 90% of the possible hybridizedcombinations of said first series, said second series, and said thirdseries can be produced in 10 hours or less.

Example 1G

A flow cytometry method for assaying a target of a sample, comprising:i) providing to the sample: 1) a molecular probe, comprising a bindingmoiety conjugated to a first oligonucleotide sequence; and 2) adetectable component, comprising a signal generating moiety conjugatedto a second oligonucleotide sequence that is complementary to the firstoligonucleotide sequence of the molecular probe; ii) binding the targetin the sample with the binding moiety of the molecular probe; iii)hybridizing the first oligonucleotide sequence of the molecular probewith the second oligonucleotide sequence of the detectable component;and iv) detecting a signal generated from the hybridized detectablecomponent using flow cytometry; wherein the method is characterized byone or more of the following: a) the conjugation between the firstoligonucleotide sequence and the binding moiety and conjugation betweenthe second oligonucleotide sequence and the signal generating moiety,comprises one or more covalent bond linkages, comprising a hydrazone,oxime, triazine, or other covalent bond, wherein the formation of theconjugates are at least 90% efficient; and b) the binding moietycomprises a strong binding affinity for the target.

Example 2G

The method of Example 1G, wherein the mode of addition comprises: i) themolecular probe and the detectable component are combined together andhybridized prior to contacting the sample; ii) the molecular probe iscombined with the sample prior to the addition of the detectablecomponent; or iii) the detectable component is combined with the sampleprior to the addition of the molecular probe.

Example 3G

The method of any one of Examples 1G-2G, wherein the method comprises:i) the molecular probe binding the target prior to hybridizing with thedetectable component; or ii) the molecular probe hybridizing with thedetectable component prior to binding the target.

Example 4G

A flow cytometry method for assaying a target of a sample, comprising:i) providing to the sample: 1) a molecular probe, comprising a bindingmoiety conjugated to a first oligonucleotide sequence; 2) a detectablecomponent, comprising a signal generating moiety conjugated to a secondoligonucleotide sequence; and 3) a universal adapter, comprising anoligonucleotide sequence having a first sequence segment complementaryto the first oligonucleotide sequence of the molecular probe and asecond sequence segment complementary to the second oligonucleotidesequence of the detectable component; ii) binding the target in thesample with the binding moiety of the molecular probe; iii) hybridizingthe first oligonucleotide sequence of the molecular probe to the firstoligonucleotide sequence segment of the universal adapter; iv)hybridizing the second oligonucleotide sequence of the detectablecomponent to the second oligonucleotide sequence segment of theuniversal adapter; and v) detecting a signal generated from thehybridized detectable component using flow cytometry; wherein the methodis characterized by one or more of the following: a) the conjugationbetween the first oligonucleotide sequence and the binding moiety andconjugation between the second complementary oligonucleotide sequenceand the signal generating moiety, comprises one or more covalent bondlinkages, comprising a hydrazone, oxime, triazine, or other covalentbond, wherein the formation of the conjugates are at least 90%efficient; and b) the binding moiety comprises a strong binding affinityfor the target.

Example 5G

The method of Example 4G, wherein the mode of addition comprises: i) themolecular probe, the universal adapter, and the detectable component arecombined together and hybridized prior to contacting the sample; ii) themolecular probe and the universal adapter are combined together andhybridized prior to contacting the sample; iii) the detectable componentand the universal adapter are combined together and hybridized prior tocontacting the sample; iv) the molecular probe, alone or in combinationwith the detectable component, is combined with the sample prior to theaddition of the universal adapter; or v) the universal adapter iscombined with the sample prior to the addition of the molecular probeand/or the detectable component.

Example 6G

The method of Example 4G or 5G, wherein the method comprises: i) themolecular probe hybridizing with the universal adapter prior to saidmolecular probe binding the target; ii) the molecular probe hybridizingwith the universal adapter after said molecular probe binds the target;iii) the detectable component hybridizing with the universal adapterprior to the molecular probe binding the target; iv) the detectablecomponent hybridizing with the universal adapter after the molecularprobe binds the target; v) the universal adapter hybridizing with themolecular probe and hybridizing with the detectable component prior tosaid molecular probe binding the target; or vi) the universal adapterhybridizing with the molecular probe and hybridizing with the detectablecomponent after said molecular probe binds the target.

Example 7G

The method of any one of Examples 1G-6G, wherein: i) the assay comprisesa singleplex or multiplex assay; and ii) the assay detects, measures, orquantifies the level of binding and/or amount of the target present inthe sample with flow cytometry.

Example 8G

The method of any one of Examples 1G-7G, wherein method furthercomprises detecting, measuring, or quantifying the level of bindingand/or amount of the target present in the sample with one or more ofthe following: immunomagnetic cellular depletion, immunomagnetic cellcapture, array, bead array, multiplex bead array, microarray, antibodyarray, cellular array, chemiluminescence, infrared, microscopy, imaging,high content screening (HCS), mass cytometry, lateral flow immunoassay,immunodetection, immunoturbidity, latex agglutination, gold particleagglutination, visual inspection, a change in light transmittancethrough said sample, increased light transmittance through said sample,immunohistochemistry (IHC), immunocytochemistry (ICC), in situhybridization (ISH), enzyme immuno-assay (EIA), enzyme linkedimmuno-assay (ELISA), ELISpot, a blotting method, a Western blot, aSouthern blot, a Southwestern blot, labeling inside an electrophoresissystem, labeling on a surface, labeling on an array, PCR amplification,elongation followed by PCR amplification, immunoprecipitation,co-immunoprecipitation, chromatin immunoprecipitation, pretargetingimaging, therapeutic agent, or combinations thereof.

Example 9G

The method of any one of Examples 1G-8G, wherein: i) the assay comprisesa singleplex or multiplex assay; and ii) the assay detects, measures, orquantifies the level of binding and/or amount of the target present inthe sample with one or more of the following: flow cytometry,microscopy, imaging, high content screening (HCS), multiplex bead array,microarray, antibody array, cellular array, immunohistochemistry (IHC),immunocytochemistry (ICC), in situ hybridization (ISH), enzymeimmuno-assay (EIA), enzyme linked immuno-assay (ELISA), ELISpot, or ablotting method.

Example 10G

The method of any one of Examples 1G-9G, wherein: i) the assay comprisesa singleplex or multiplex assay; and ii) the assay detects, measures, orquantifies the level of binding and/or amount of the target present inthe sample with one or more of the following: flow cytometry, masscytometry, lateral flow immunoassay, immunohistochemistry (IHC),immunocytochemistry (ICC), immunoprecipitation, pretargeting imaging,therapeutic agent, or combinations thereof.

Example 11G

The method of any one of Examples 1G-10G, wherein the sample ischaracterized as at least one or more of the following: i) a complexsample; and ii) a homogeneous or a heterogeneous mixture; wherein saidsample comprises at least one or more of the following: a) one or moreanalytes having substantially the same or substantially differentbinding specificities; b) one or more of the following biologiccomponents, comprising: cells, membranes, biological molecules,metabolites, or disease biomarkers; and c) a biological fluid or afluidized biological tissue.

Example 12G

The method of any one of Examples 1G-11G, wherein the hybridizationefficiency of the first oligonucleotide sequence to the secondoligonucleotide sequence is at least 50% with respect to the firstdetectable component, under the hybridization conditions employed.

Example 13G

The method of any one of Examples 1G-12G, wherein the molecular probecomprises one or more of the following properties: i) a molecular weightof between about 15,000 Daltons to about 450,000 Daltons; ii) asolubility that is substantially the same as that of the unconjugatedbinding moiety; iii) a solubility that minimizes non-specific binding tothe target; iv) the first oligonucleotide sequence of the molecularprobe does not adversely affect the solubility of the binding moiety; v)interacts and binds to the target via interactions other thanexclusively electrostatic; vi) a unique, distinguishable, and/orspecifically designed oligonucleotide sequence; and vii) the firstoligonucleotide sequence of the molecular probe is uniquely andspecifically designed to hybridize to the second oligonucleotidesequence of the detectable component.

Example 14G

The method of any one of Examples 1G-13G, wherein the method ofdetection generates less false positives than secondary antibodydetection methods.

Example 15G

The method of any one of Examples 1G-14G, wherein the method furthercomprises: i) preparing the molecular probe; and ii) preparing thedetectable component; wherein the prepared molecular probe and prepareddetectable component have at least 90% purity.

Example 16G

The method of any one of Examples 1G-15G, wherein the method furthercomprises preparing and isolating the molecular probe, comprising: i)providing the binding moiety; ii) conjugating the binding moiety with atleast one first oligonucleotide sequence at greater than 90% efficiencyto form binding moiety-oligonucleotide conjugates; and iii) isolatingthe binding moiety-oligonucleotide conjugates from the conjugationmixture by binding, retaining, and/or retarding a substantial portionof: a) the conjugates, removing a substantial portion of theunconjugated first oligonucleotide sequence in a wash step followed byrelease of the bound, retained, and/or retarded conjugates; or b) theunconjugated first oligonucleotide sequences, followed by collecting asubstantial portion of the non-bound, non-retained, and/or non-retardedconjugates in a wash step.

Example 17G

The method of any one of Examples 1G-16G, wherein the method furthercomprises preparing and isolating the detectable component, comprising:i) providing a plurality of the signal generating moiety; ii)conjugating the second oligonucleotide sequence with at least one of theplurality of the signal generating moiety at greater than 90% efficiencyto form signal generating moiety-second oligonucleotide conjugates; andiii) isolating the signal generating moiety-second oligonucleotideconjugates from the conjugation mixture by binding, retaining, and/orretarded a substantial portion of: a) the conjugates, removing asubstantial portion of the unconjugated second oligonucleotide sequencesin a wash step followed by release of the bound, retained, and/orretarded conjugates; or b) the unconjugated second oligonucleotidesequences, followed by collecting a substantial portion of thenon-bound, non-retained, and/or non-retarded conjugates in a wash step.

Example 18G

The method of any one of Examples 1G-17G, wherein the detectablecomponent comprises a scaffold conjugated to the second oligonucleotidesequence, and wherein said scaffold comprises one or more signalgenerating moieties.

Example 19G

The method of Example 18G, wherein the scaffold comprises a dendrimer, apolysaccharide, a dextran, a protein, a peptide, a furtheroligonucleotide sequence, a portion of the second oligonucleotidesequence that is not complementary to the first oligonucleotide sequenceof the molecular probe, a polymer, a hydrophilic polymer, a bead, ananoparticle, or combinations or derivatives thereof.

Example 20G

The method of any one of Examples 1G-19G, wherein the method furthercomprises preparing and isolating a detectable component comprising ascaffold conjugated to the second oligonucleotide sequence, wherein thescaffold comprises one or more signal generating moieties, said methodcomprising: i) providing a plurality of the scaffolds comprising the oneor more signal generating moieties; ii) conjugating the secondoligonucleotide sequence with at least one of the plurality of scaffoldsat greater than 90% efficiency to form scaffold-second oligonucleotideconjugates; and iii) isolating the scaffold-second oligonucleotideconjugates from the conjugation mixture by binding, retaining, and/orretarding a substantial portion of: a) the conjugates, removing asubstantial portion of the unconjugated second oligonucleotide sequencesin a wash step followed by release of the bound, retained, and/orretarded conjugates; or b) the unconjugated second oligonucleotidesequences, followed by collecting a substantial portion of thenon-bound, non-retained, and/or non-retarded conjugates in a wash step.

Example 21G

The method of any one of Examples 16G-20G, wherein the isolation steputilizes an immobilized binder, chromatography, affinity chromatography,size exclusion chromatography, HPLC, reverse-phase chromatography,electrophoresis, capillary electrophoresis, polyacrylamide gelelectrophoresis, agarose gel electrophoresis, free flow electrophoresis,differential centrifugation, thin layer chromatography,immunoprecipitation, hybridization, solvent extraction, dialysis,filtration, diafiltration, tangential flow filtration, ion exchangechromatography, hydrophobic interaction chromatography, or combinationsthereof.

Example 22G

The method of any one of Examples 16G-20G, wherein the isolation steputilizes an immobilized binder, affinity chromatography, size exclusionchromatography, electrophoresis, differential centrifugation,immunoprecipitation, hybridization, solvent extraction, dialysis,filtration, diafiltration, ion exchange chromatography, hydrophobicinteraction chromatography, or combinations thereof.

Example 23G

The method of any one of Examples 16G-20G, wherein the isolation steputilizes an immobilized binder, affinity chromatography, size exclusionchromatography, differential centrifugation, dialysis, filtration,hydrophobic interaction chromatography, or combinations thereof.

Example 24G

The method of any one of Examples 1G-23G, wherein the binding moietycomprises an antibody, a monoclonal antibody, a polyclonal antibody, anenzyme, a protein, a peptide, a carbohydrate, a nuclear receptor, asmall molecule, an aptamer, a chelator, or combinations or derivativesthereof.

Example 25G

The method of any one of Examples 1G-24G, wherein the sample comprisesone or more targets.

Example 26G

The method of any one of Examples 1G-25G, wherein the target is abiological target.

Example 27G

The method of Example 26G, wherein the biological target comprises anantigen, a pathogen, a protein, a peptide, an epitope, acarbohydrate-containing molecule, a small molecule, or combinations orderivatives thereof.

Example 28G

The method of any one of Examples 1G-27G, wherein the signal generatingmoiety or the one or more signal generating moieties of the detectablecomponent or the hybridized detectable component, comprises one or moreof the following: a directly detectable signal generating moiety, anindirectly detectable signal generating moiety, a fluorescent dye, afluorophore, a fluorochrome, a chromophore, a biofluorescent protein, aluminescent species, a chemiluminescent compound, aelectrochemiluminescent label, a bioluminescent label, a phosphorescentspecies, a fluorophore labeled DNA dendrimer, Quantum Dot, a tandem dye,a FRET dye, a heavy atom, a spin label, a radioactive isotope, ananoparticle, a light scattering nanoparticle or microsphere, adiffracting particle, a polymer, a polymer particle, a bead, a solidsurface, a Raman particle, a metal particle, a stable isotope, a heavymetal chelate, a magnetic particle, an RFID tag, a microbarcodeparticle, an enzyme, an enzyme substrate, a molecule specificallyrecognized by another substance carrying a label or reacts with asubstance carrying a label, an antibody, an antibody fragment, anantigen, a nucleic acid, a nucleic acid analog, oligonucleotide,oligonucleotide analog, complementary oligonucleotide, complementaryoligonucleotide analog, a ligand, a protein, a peptide ligand, a proteinsubstrate, a receptor; a substrate, a secondary reporter, a hapten, orcombinations or derivatives thereof.

Example 29G

The method of any one of Examples 1G-28G, wherein the one or more signalgenerating moieties provides an enhanced signal that minimizes detectionerrors from background noise, relative to conventionally labeled bindingmoieties.

Example 30G

The method of any one of Examples 1G-29G, wherein the molecular probe,the detectable component, and/or universal adapter further comprises aspacer group, comprising a polymerized ethylene oxide, a PEG, a PEO, aprotein, a peptide, a DNA, an RNA, an oligonucleotide sequence, or adextran.

Example 31G

The method of any one of Examples 1G-30G, wherein the binding moiety,the scaffold, the first oligonucleotide sequence, and/or the secondoligonucleotide sequence comprise HyNic or 4-FB.

Example 32G

The method of any one of Examples 1G-31G, wherein the detectablecomponent comprises a unique, distinguishable, and/or specificallydesigned second oligonucleotide sequence.

Example 33G

The method of any one of Examples 1G-32G, wherein the firstoligonucleotide sequence, second oligonucleotide sequence, and/oroligonucleotide sequence segment comprising an oligonucleotide sequenceconjugated at the 3′-position, an oligonucleotide sequence conjugated atthe 5′-position, linear oligonucleotide sequences, branchedoligonucleotide sequences, LNAs, PNAs, oligonucleotide sequencesoptionally covalently attached to other moieties, or combinations orderivatives thereof.

Example 34G

The method of any one of Examples 1G-33G, wherein the sample comprisesone or more targets comprising at least a first target and at least asecond target.

Example 35G

The method of any one of Examples 1G-34G, wherein a plurality ofmolecular probes and a plurality of detectable components are providedto the sample.

Example 36G

The method of any one of Examples 1G-35G, wherein a plurality ofuniversal adapters are provided to the sample.

Example 37G

The method of any one of Examples 1G-36G, wherein the binding affinityfor the target is 10⁻⁴ M or less.

Example 38G

The method of any one of Examples 1G-37G, wherein the binding affinityfor the at least first target is 10⁻⁴ M or less.

Example 39G

The method of any one of Examples 1G-38G, wherein the binding affinityfor the at least second target is 10⁻⁴ M or less.

Example 40G

The method of any one of Examples 1G-39G, wherein the method comprisesan automated system or robotic system.

Example 41G

The method of any one of Examples 1G-41G, wherein the method furthercomprises removing the hybridized detectable component or plurality ofdetectable components from the bound target or plurality of targets,respectively, wherein said removal is by a washing or stripping process.

Example 41G

The method of Example 41G, wherein the removal comprises de-hybridizingthe detectable component or the plurality of detectable components,respectively.

Example 42G

The method of Examples 40G or 41G, wherein the method further comprisesre-probing with a second detectable component or second plurality ofdetectable components, respectively, wherein said second detectablecomponent comprises at least one second signal generating moietiesconjugated to a second oligonucleotide sequence or a complementarysecond oligonucleotide sequence, or said second plurality of detectablecomponents are prepared by independently pairing, via conjugation, asecond plurality of signal generating moieties and a second plurality ofsecond oligonucleotide sequences or a second plurality of complementarysecond oligonucleotide sequences.

Example 43G

A flow cytometry method for assaying one or more targets of a sample,comprising: i) providing to the sample: 1) a plurality of molecularprobes, comprising: A) at least a first molecular probe having a firstbinding moiety conjugated to a first oligonucleotide sequence; and B) atleast a second molecular probe having a second binding moiety conjugatedto a second oligonucleotide sequence; and 2) a plurality of detectablecomponents, comprising: A) at least a first detectable component havinga first signal generating moiety conjugated to a first complementaryoligonucleotide sequence; and B) at least a second detectable componenthaving a second signal generating moiety conjugated to a secondcomplementary oligonucleotide sequence; ii) binding the one or moretargets, comprising at least one of the following: 1) binding at least afirst target of the one or more targets in the sample with the firstbinding moiety of the at least first molecular probe; and 2) binding atleast a second target of the one or more targets in the sample with thesecond binding moiety of the at least second molecular probe; iii)hybridizing the plurality of molecular probes and the plurality ofdetectable components, comprising at least one of the following: 1)hybridizing the first oligonucleotide sequence of at least firstmolecular probe to the first complementary oligonucleotide sequencesegment of the at least first detectable component; and 2) hybridizingthe second oligonucleotide sequence of at least second molecular probeto the second complementary oligonucleotide sequence segment of the atleast second detectable component; and iv) detecting, using flowcytometry, one or more signals generated from at least one of thefollowing: 1) the at least first hybridized detectable component; and 2)the at least second hybridized detectable component; wherein the methodis characterized by one or more of the following: a) the conjugationbetween the first oligonucleotide sequence and the first binding moiety,between the second oligonucleotide sequence and the second bindingmoiety, between the first complementary oligonucleotide sequence and thefirst signal generating moiety, and between the second complementaryoligonucleotide sequence and the second signal generating moiety,comprises one or more covalent bond linkages, comprising a hydrazone,oxime, triazine, or other covalent bond, wherein the formation of theconjugates are at least 90% efficient; and b) the first binding moietycomprises a strong binding affinity for the at least first target of theone or more targets and the second binding moiety comprises a strongbinding affinity for the at least second target of the one or moretargets.

Example 44G

A flow cytometry method for assaying one or more targets of a sample,comprising: i) providing to the sample: a) a plurality of molecularprobes, comprising: a first oligonucleotide sequence independentlypaired, via conjugation, to a plurality of binding moieties comprisingat least a first binding moiety and at least a second binding moiety; b)a plurality of detectable components, comprising: a plurality of secondoligonucleotide sequences independently paired, via conjugation, to aplurality of one or more signal generating moieties comprising at leasta first signal generating moiety and at least a second signal generatingmoiety; and c) a plurality of universal adapters, comprising: a firstoligonucleotide sequence segment, complementary to the firstoligonucleotide sequence of said plurality of molecular probes,independently paired with a plurality of second oligonucleotide sequencesegments complementary to the plurality of second oligonucleotidesequences of said plurality of detectable components; ii) binding theone or more targets, comprising at least one of the following: a)binding at least a first target of the one or more targets in the samplewith the first binding moiety of the at least first molecular probe; andb) binding at least a second target of the one or more targets in thesample with the second binding moiety of the at least second molecularprobe; iii) hybridizing the plurality of molecular probes and theplurality of detectable components with the plurality of universaladapters; and iv) detecting, using flow cytometry, one or more signalsgenerated from at least one of the following: a) the at least firsthybridized detectable component; and b) the at least second hybridizeddetectable component; wherein the method is characterized by one or moreof the following: A) the conjugation between the first oligonucleotidesequence and the first binding moiety, between the secondoligonucleotide sequence and the second binding moiety, between thefirst complementary oligonucleotide sequence and the first signalgenerating moiety, and between the second complementary oligonucleotidesequence and the second signal generating moiety, comprises one or morecovalent bond linkages, comprising a hydrazone, oxime, triazine, orother covalent bond, wherein the formation of the conjugates are atleast 90% efficient; and B) the first binding moiety comprises a strongbinding affinity for the at least first target of the one or moretargets and the second binding moiety comprises a strong bindingaffinity for the at least second target of the one or more targets.

Example 1H

A method for assaying a target of a sample, comprising: i) providing tothe sample: 1) a molecular probe, comprising a binding moiety conjugatedto a first oligonucleotide sequence; and 2) a detectable component,comprising a signal generating moiety conjugated to a secondoligonucleotide sequence that is complementary to the firstoligonucleotide sequence of the molecular probe; ii) binding the targetin the sample with the binding moiety of the molecular probe; iii)hybridizing the oligonucleotide sequence of the molecular probe with thesecond oligonucleotide sequence of the detectable component; and iv)detecting a signal generated from the hybridized detectable componentusing one or more of the following systems: microscopy, imaging, highcontent screening (HCS), mass cytometry, lateral flow immunoassay,immunodetection, immunohistochemistry (IHC), immunocytochemistry (ICC),or combinations thereof; wherein the method is characterized by one ormore of the following: a) the conjugation between the firstoligonucleotide sequence and the binding moiety and conjugation betweenthe second oligonucleotide sequence and the signal generating moiety,comprises one or more covalent bond linkages, comprising a hydrazone,oxime, triazine, or other covalent bond, wherein the formation of theconjugates are at least 90% efficient; and b) the binding moietycomprises a strong binding affinity for the target.

Example 2H

The method of Example 1H, wherein the mode of addition comprises: i) themolecular probe and the detectable component are combined together andhybridized prior to contacting the sample; ii) the molecular probe iscombined with the sample prior to the addition of the detectablecomponent; or iii) the detectable component is combined with the sampleprior to the addition of the molecular probe.

Example 3H

The method of any one of Examples 1H-2H, wherein the method comprises:i) the molecular probe binding the target prior to hybridizing with thedetectable component; or ii) the molecular probe hybridizing with thedetectable component prior to binding the target.

Example 4H

The method of any one of Examples 1H-3H, wherein: i) the assay comprisesa singleplex or multiplex assay; and ii) the assay detects, measures, orquantifies the level of binding and/or amount of the target present inthe sample with the one or more of following systems: microscopy,imaging, high content screening (HCS), mass cytometry, lateral flowimmunoassay, immunodetection, immunohistochemistry (IHC),immunocytochemistry (ICC), or combinations thereof.

Example 5H

A method for assaying a target of a sample, comprising: i) providing tothe sample: 1) a molecular probe, comprising a binding moiety conjugatedto a first oligonucleotide sequence; 2) a detectable component,comprising a signal generating moiety conjugated to a secondoligonucleotide sequence; and 3) a universal adapter, comprising anoligonucleotide sequence having a first sequence segment complementaryto the first oligonucleotide sequence of the molecular probe and asecond sequence segment complementary to the second oligonucleotidesequence of the detectable component; ii) binding the target in thesample with the binding moiety of the molecular probe; iii) hybridizingthe first oligonucleotide sequence of the molecular probe to the firstoligonucleotide sequence segment of the universal adapter; iv)hybridizing the second oligonucleotide sequence of the detectablecomponent to the second oligonucleotide sequence segment of theuniversal adapter; and v) detecting a signal generated from thehybridized detectable component using one or more of the followingsystems: microscopy, imaging, high content screening (HCS), masscytometry, lateral flow immunoassay, immunodetection,immunohistochemistry (IHC), immunocytochemistry (ICC), or combinationsthereof; wherein the method is characterized by one or more of thefollowing: a) the conjugation between the first oligonucleotide sequenceand the binding moiety and conjugation between the second complementaryoligonucleotide sequence and the signal generating moiety, comprises oneor more covalent bond linkages, comprising a hydrazone, oxime, triazine,or other covalent bond, wherein the formation of the conjugates are atleast 90% efficient; and b) the binding moiety comprises a strongbinding affinity for the target.

Example 6H

The method of Example 5H, wherein the mode of addition comprises: i) themolecular probe, the universal adapter, and the detectable component arecombined together and hybridized prior to contacting the sample; ii) themolecular probe and the universal adapter are combined together andhybridized prior to contacting the sample; iii) the detectable componentand the universal adapter are combined together and hybridized prior tocontacting the sample; iv) the molecular probe, alone or in combinationwith the detectable component, is combined with the sample prior to theaddition of the universal adapter; or v) the universal adapter iscombined with the sample prior to the addition of the molecular probeand/or the detectable component.

Example 7H

The method of Examples 5H or 6H, wherein the method comprises: i) themolecular probe hybridizing with the universal adapter prior to saidmolecular probe binding the target; ii) the molecular probe hybridizingwith the universal adapter after said molecular probe binds the target;iii) the detectable component hybridizing with the universal adapterprior to the molecular probe binding the target; iv) the detectablecomponent hybridizing with the universal adapter after the molecularprobe binds the target; v) the universal adapter hybridizing with themolecular probe and hybridizing with the detectable component prior tosaid molecular probe binding the target; or vi) the universal adapterhybridizing with the molecular probe and hybridizing with the detectablecomponent after said molecular probe binds the target.

Example 8H

The method of any one of Examples 1H-7H, wherein method furthercomprises detecting, measuring, or quantifying the level of bindingand/or amount of the target present in the sample with one or more ofthe following: flow cytometry, immunomagnetic cellular depletion,immunomagnetic cell capture, array, bead array, multiplex bead array,microarray, antibody array, cellular array, chemiluminescence, infrared,immunoturbidity, latex agglutination, gold particle agglutination,visual inspection, a change in light transmittance through said sample,increased light transmittance through said sample, in situ hybridization(ISH), enzyme immuno-assay (EIA), enzyme linked immuno-assay (ELISA),ELISpot, a blotting method, a Western blot, a Southern blot, aSouthwestern blot, labeling inside an electrophoresis system, labelingon a surface, labeling on an array, PCR amplification, elongationfollowed by PCR amplification, immunoprecipitation,co-immunoprecipitation, chromatin immunoprecipitation, pretargetingimaging, therapeutic agent, or combinations thereof.

Example 9H

The method of any one of Examples 1H-8H, wherein the method carries outthe detecting, measuring, or quantifying via microscopy, imaging, highcontent screening (HCS), or combinations thereof.

Example 10H

The method of any one of Examples 1H-9H, wherein the method carries outthe detecting, measuring, or quantifying via mass cytometry, lateralflow immunoassay, immunodetection, immunohistochemistry (IHC),immunocytochemistry (ICC), or combinations thereof.

Example 11H

The method of any one of Examples 1H-10H, wherein the sample ischaracterized as at least one or more of the following: i) a complexsample; and ii) a homogeneous or a heterogeneous mixture; wherein saidsample comprises at least one or more of the following: a) one or moreanalytes having substantially the same or substantially differentbinding specificities; b) one or more of the following biologiccomponents, comprising: cells, membranes, biological molecules,metabolites, or disease biomarkers; and c) a biological fluid or afluidized biological tissue.

Example 12H

The method of any one of Examples 1H-11H, wherein the hybridizationefficiency of the oligonucleotide sequence to the second oligonucleotidesequence is at least 50% with respect to the detectable component, underthe hybridization conditions employed.

Example 13H

The method of any one of Examples 1H-12H, wherein the molecular probecomprises one or more of the following properties: i) a molecular weightof between about 15,000 Daltons to about 450,000 Daltons; ii) asolubility that is substantially the same as that of the unconjugatedbinding moiety; iii) a solubility that minimizes non-specific binding tothe target; iv) the oligonucleotide sequence of the molecular probe doesnot adversely affect the solubility of the binding moiety; v) interactsand binds to the target via interactions other than exclusivelyelectrostatic; vi) a unique, distinguishable, and/or specificallydesigned oligonucleotide sequence; and vii) the first oligonucleotidesequence of the molecular probe is uniquely and specifically designed tohybridize to the second oligonucleotide sequence of the detectablecomponent or to the complementary first oligonucleotide sequence segmentof the universal adapter.

Example 14H

The method of any one of Examples 1H-13H, wherein the method ofdetection generates less false positives than secondary antibodydetection methods.

Example 15H

The method of any one of Examples 1H-14H, wherein the method furthercomprises: i) preparing the molecular probe; and ii) preparing thedetectable component; wherein the prepared molecular probe and prepareddetectable component have at least 90% purity.

Example 16H

The method of any one of Examples 1H-15H, wherein the method furthercomprises preparing and isolating the molecular probe, comprising: i)providing the binding moiety; ii) conjugating the binding moiety with atleast one first oligonucleotide sequence at greater than 90% efficiencyto form binding moiety-oligonucleotide conjugates; and iii) isolatingthe binding moiety-oligonucleotide conjugates from the conjugationmixture by binding, retaining, and/or retarding a substantial portionof: a) the conjugates, removing a substantial portion of theunconjugated first oligonucleotide sequence in a wash step followed byrelease of the bound, retained, and/or retarded conjugates; or b) theunconjugated first oligonucleotide sequences, followed by collecting asubstantial portion of the non-bound, non-retained, and/or non-retardedconjugates in a wash step.

Example 17H

The method of any one of Examples 1H-16H, wherein the method furthercomprises preparing and isolating the detectable component, comprising:i) providing a plurality of the signal generating moiety; ii)conjugating the second oligonucleotide sequence with at least one of theplurality of the signal generating moiety at greater than 90% efficiencyto form signal generating moiety-second oligonucleotide conjugates; andiii) isolating the signal generating moiety-second oligonucleotideconjugates from the conjugation mixture by binding, retaining, and/orretarding a substantial portion of: a) the conjugates, removing asubstantial portion of the unconjugated second oligonucleotide sequencesin a wash step followed by release of the bound, retained, and/orretarded conjugates; or b) the unconjugated second oligonucleotidesequences, followed by collecting a substantial portion of thenon-bound, non-retained, and/or non-retarded conjugates in a wash step.

Example 18H

The method of any one of Examples 1H-17H, wherein the detectablecomponent comprises a scaffold conjugated to the second oligonucleotidesequence, and wherein said scaffold comprises one or more signalgenerating moieties.

Example 19H

The method of Example 18H, wherein the scaffold comprises a dendrimer, apolysaccharide, a dextran, a protein, a peptide, a furtheroligonucleotide sequence, a portion of the second oligonucleotidesequence that is not complementary to the first oligonucleotide sequenceof the molecular probe, a polymer, a hydrophilic polymer, a bead, ananoparticle, or combinations or derivatives thereof.

Example 20H

The method of any one of Examples 1H-19H, wherein the method furthercomprises preparing and isolating a detectable component comprising ascaffold conjugated to the second oligonucleotide sequence, wherein thescaffold comprises one or more signal generating moieties, said methodcomprising: i) providing a plurality of the scaffolds comprising the oneor more signal generating moieties; ii) conjugating the secondoligonucleotide sequence with at least one of the plurality of scaffoldsat greater than 90% efficiency to form scaffold-second oligonucleotideconjugates; and iii) isolating the scaffold-second oligonucleotideconjugates from the conjugation mixture by binding, retaining, and/orretarding a substantial portion of: a) the conjugates, removing asubstantial portion of the unconjugated second oligonucleotide sequencesin a wash step followed by release of the bound, retained, and/orretarded conjugates; or b) the unconjugated second oligonucleotidesequences, followed by collecting a substantial portion of thenon-bound, non-retained, and/or non-retarded conjugates in a wash step.

Example 21H

The method of any one of Examples 16-20H, wherein the isolation steputilizes an immobilized binder, affinity chromatography, size exclusionchromatography, HPLC, reverse-phase chromatography, electrophoresis,capillary electrophoresis, polyacrylamide gel electrophoresis, agarosegel electrophoresis, free flow electrophoresis, differentialcentrifugation, thin layer chromatography, immunoprecipitation,hybridization, solvent extraction, dialysis, filtration, diafiltration,tangential flow filtration, ion exchange chromatography, hydrophobicinteraction chromatography, or combinations thereof.

Example 22H

The method of any one of Examples 16H-20H, wherein the isolation steputilizes an immobilized binder, affinity chromatography, size exclusionchromatography, electrophoresis, differential centrifugation,immunoprecipitation, hybridization, solvent extraction, dialysis,filtration, diafiltration, ion exchange chromatography, hydrophobicinteraction chromatography, or combinations thereof.

Example 23H

The method of any one of Examples 16H-20H, wherein the isolation steputilizes an immobilized binder, affinity chromatography, size exclusionchromatography, differential centrifugation, dialysis, filtration,hydrophobic interaction chromatography, or combinations thereof.

Example 24H

The method of any one of Examples 1H-23H, wherein the binding moietycomprises an antibody, a monoclonal antibody, a polyclonal antibody, anenzyme, a protein, a peptide, a carbohydrate, a nuclear receptor, asmall molecule, an aptamer, a chelator, or combinations or derivativesthereof.

Example 25H

The method of any one of Examples 1H-24H, wherein the sample comprisesone or more targets.

Example 26H

The method of any one of Examples 1H-25H, wherein the target is abiological target.

Example 27H

The method of Example 26H, wherein the biological target comprises anantigen, a pathogen, a protein, a peptide, an epitope, acarbohydrate-containing molecule, a small molecule, or combinations orderivatives thereof.

Example 28H

The method of any one of Examples 1H-27H, wherein the signal generatingmoiety or the one or more signal generating moieties of the detectablecomponent or the hybridized detectable component, comprises one or moreof the following: a directly detectable signal generating moiety, anindirectly detectable signal generating moiety, a fluorescent dye, afluorophore, a fluorochrome, a chromophore, a biofluorescent protein, aluminescent species, a chemiluminescent compound, aelectrochemiluminescent label, a bioluminescent label, a phosphorescentspecies, a fluorophore labeled DNA dendrimer, Quantum Dot, a tandem dye,a FRET dye, a heavy atom, a spin label, a radioactive isotope, ananoparticle, a light scattering nanoparticle or microsphere, adiffracting particle, a polymer, a polymer particle, a bead, a solidsurface, a Raman particle, a metal particle, a stable isotope, a heavymetal chelate, a magnetic particle, an RFID tag, a microbarcodeparticle, an enzyme, an enzyme substrate, a molecule specificallyrecognized by another substance carrying a label or reacts with asubstance carrying a label, an antibody, an antibody fragment, anantigen, a nucleic acid, a nucleic acid analog, oligonucleotide,oligonucleotide analog, complementary oligonucleotide, complementaryoligonucleotide analog, a ligand, a protein, a peptide ligand, a proteinsubstrate, a receptor; a substrate, a secondary reporter, a hapten, orcombinations or derivatives thereof.

Example 29H

The method of any one of Examples 1H-28H, wherein the one or more signalgenerating moieties provides an enhanced signal that minimizes detectionerrors from background noise, relative to conventionally labeled bindingmoieties.

Example 30H

The method of any one of Examples 1H-29H, wherein the molecular probe,the detectable component, and/or universal adapter further comprises aspacer group, comprising a polymerized ethylene oxide, a PEG, a PEO, aprotein, a peptide, a DNA, an RNA, an oligonucleotide sequence, or adextran.

Example 31H

The method of any one of Examples 1H-30H, wherein the binding moiety,the scaffold, the oligonucleotide sequence, and/or the complementaryoligonucleotide sequence comprise HyNic or 4-FB.

Example 32H

The method of any one of Examples 1H-31H, wherein the detectablecomponent comprises a unique, distinguishable, and/or specificallydesigned complementary oligonucleotide sequence.

Example 33H

The method of any one of Examples 1H-32H, wherein the oligonucleotidesequence, complementary oligonucleotide sequence, and/or oligonucleotidesequence segment comprises an oligonucleotide sequence conjugated at the3′-position, an oligonucleotide sequence conjugated at the 5′-position,linear oligonucleotide sequences, branched oligonucleotide sequences,LNAs, PNAs, oligonucleotide sequences optionally covalently attached toother moieties, or combinations or derivatives thereof.

Example 34H

The method of any one of Examples 1H-33H, wherein the sample comprisesone or more targets.

Example 35H

The method of any one of Examples 1H-34H, wherein a plurality ofmolecular probes and a plurality of detectable components are providedto the sample.

Example 36H

The method of any one of Examples 1H-35H, wherein a plurality ofuniversal adapters are provided to the sample.

Example 37H

The method of any one of Examples 1H-36H, wherein the binding affinityfor the target is 10⁻⁴ M or less.

Example 38H

The method of any one of Examples 1H-37H, wherein the binding affinityfor the at least first target is 10⁻⁴M or less.

Example 39H

The method of any one of Examples 1H-38H, wherein the binding affinityfor the at least second target is 10⁻⁴ M or less.

Example 40H

The method of any one of Examples 1H-39H, wherein the method comprisesan automated system or robotic system.

Example 41H

The method of any one of Examples 1H-41H, wherein the method furthercomprises removing the hybridized detectable component or plurality ofdetectable components from the bound target or plurality of targets,respectively, wherein said removal is by a washing or stripping process.

Example 41H

The method of Example 41H, wherein the removal comprises de-hybridizingthe detectable component or the plurality of detectable components,respectively.

Example 42H

The method of Examples 40H or 41H, wherein the method further comprisesre-probing with a second detectable component or second plurality ofdetectable components, respectively, wherein said second detectablecomponent comprises at least one second signal generating moietiesconjugated to a second oligonucleotide sequence or a complementarysecond oligonucleotide sequence, or said second plurality of detectablecomponents are prepared by independently pairing, via conjugation, asecond plurality of signal generating moieties and a second plurality ofsecond oligonucleotide sequences or a second plurality of complementarysecond oligonucleotide sequences.

Example 43H

A method for assaying one or more targets of a sample, comprising: i)providing to the sample: 1) a plurality of molecular probes, comprising:A) at least a first molecular probe having a first binding moietyconjugated to a first oligonucleotide sequence; and B) at least a secondmolecular probe having a second binding moiety conjugated to a secondoligonucleotide sequence; and 2) a plurality of detectable components,comprising: A) at least a first detectable component having a firstsignal generating moiety conjugated to a first complementaryoligonucleotide sequence; and B) at least a second detectable componenthaving a second signal generating moiety conjugated to a secondcomplementary oligonucleotide sequence; ii) binding the one or moretargets, comprising at least one of the following: 1) binding at least afirst target of the one or more targets in the sample with the firstbinding moiety of the at least first molecular probe; and 2) binding atleast a second target of the one or more targets in the sample with thesecond binding moiety of the at least second molecular probe; iii)hybridizing the plurality of molecular probes and the plurality ofdetectable components, comprising at least one of the following: 1)hybridizing the first oligonucleotide sequence of at least firstmolecular probe to the first complementary oligonucleotide sequencesegment of the at least first detectable component; and 2) hybridizingthe second oligonucleotide sequence of at least second molecular probeto the second complementary oligonucleotide sequence segment of the atleast second detectable component; and iv) detecting one or more signalsgenerated from the at least first hybridized detectable component and/orthe at least second hybridized detectable component using one or more ofthe following systems: microscopy, imaging, high content screening(HCS), mass cytometry, lateral flow immunoassay, immunodetection,immunohistochemistry (IHC), immunocytochemistry (ICC), or combinationsthereof; wherein the method is characterized by one or more of thefollowing: a) the conjugation between the first oligonucleotide sequenceand the first binding moiety, between the second oligonucleotidesequence and the second binding moiety, between the first complementaryoligonucleotide sequence and the first signal generating moiety, andbetween the second complementary oligonucleotide sequence and the secondsignal generating moiety, comprises one or more covalent bond linkages,comprising a hydrazone, oxime, triazine, or other covalent bond, whereinthe formation of the conjugates are at least 90% efficient; and b) thefirst binding moiety comprises a strong binding affinity for the atleast first target of the one or more targets and the second bindingmoiety comprises a strong binding affinity for the at least secondtarget of the one or more targets.

Example 44H

A method for assaying one or more targets of a sample, comprising: i)providing to the sample: a) a plurality of molecular probes, comprising:a first oligonucleotide sequence independently paired, via conjugation,to a plurality of binding moieties comprising at least a first bindingmoiety and at least a second binding moiety; b) a plurality ofdetectable components, comprising: a plurality of second oligonucleotidesequences independently paired, via conjugation, to a plurality of oneor more signal generating moieties comprising at least a first signalgenerating moiety and at least a second signal generating moiety; and c)a plurality of universal adapters, comprising: a first oligonucleotidesequence segment, complementary to the first oligonucleotide sequence ofsaid plurality of molecular probes, independently paired with aplurality of second oligonucleotide sequence segments complementary tothe plurality of second oligonucleotide sequences of said plurality ofdetectable components; ii) binding the one or more targets, comprisingat least one of the following: a) binding at least a first target of theone or more targets in the sample with the first binding moiety of theat least first molecular probe; and b) binding at least a second targetof the one or more targets in the sample with the second binding moietyof the at least second molecular probe; iii) hybridizing the pluralityof molecular probes and the plurality of detectable components with theplurality of universal adapters; and iv) detecting one or more signalsgenerated from the at least first hybridized detectable component and/orthe at least second hybridized detectable component using one or more ofthe following systems: microscopy, imaging, high content screening(HCS), mass cytometry, lateral flow immunoassay, immunodetection,immunohistochemistry (IHC), immunocytochemistry (ICC), or combinationsthereof; wherein the method is characterized by one or more of thefollowing: A) the conjugation between the first oligonucleotide sequenceand the first binding moiety, between the second oligonucleotidesequence and the second binding moiety, between the first complementaryoligonucleotide sequence and the first signal generating moiety, andbetween the second complementary oligonucleotide sequence and the secondsignal generating moiety, comprises one or more covalent bond linkages,comprising a hydrazone, oxime, triazine, or other covalent bond, whereinthe formation of the conjugates are at least 90% efficient; and B) thefirst binding moiety comprises a strong binding affinity for the atleast first target of the one or more targets and the second bindingmoiety comprises a strong binding affinity for the at least secondtarget of the one or more targets.

Example 1I

A panel development system, comprising: i) a first panel comprising aplurality of molecular probes, said plurality of molecular probesprepared by independently pairing, via conjugation, a plurality ofbinding moieties and a plurality of oligonucleotide sequences; ii) asecond panel comprising a plurality of detectable components, saidplurality of detectable components prepared by independently pairing,via conjugation, a plurality of signal generating moieties and aplurality of complementary oligonucleotide sequences; iii) contacting asample comprising a plurality of targets with the first panel and thesecond panel, wherein: a) at least a first target having one or moredistinct bindings sites is bound by one or more molecular probes fromthe first panel; and b) one or more detectable components from thesecond panel independently hybridize with the one or more molecularprobes bound to the at least first target; iv) detecting the presence ofthe at least first hybridized-target in said sample; v) determining acharacteristic panel of molecular probes and detectable components fromsaid first and second panels that identifies the at least first targetamong the plurality of targets in said sample.

Example 2I

The panel development system of Example 1I, wherein the plurality ofoligonucleotide sequences in said first panel comprises between 2-40different oligonucleotide sequences among the plurality ofoligonucleotide sequences.

Example 3I

The panel development system of Example 1I or 2I, wherein the pluralityof complementary oligonucleotide sequences in said second panelcomprises between 2-40 different complementary oligonucleotide sequencesamong the plurality of complementary oligonucleotide sequences.

Example 4I

The panel development system of any one of Examples 1I-3I, wherein thepanel development system further comprises a re-probe panel comprising asecond plurality of detectable components, said second plurality ofdetectable components prepared by independently pairing, viaconjugation, a second plurality of signal generating moieties and asecond plurality of complementary oligonucleotide sequences.

Example 5I

A panel development system, comprising: i) a first panel comprising aplurality of molecular probes, said plurality of molecular probesprepared by independently pairing, via conjugation, a plurality ofbinding moieties and a plurality of first oligonucleotide sequences; ii)a second panel comprising a plurality of detectable components, saidplurality of detectable components prepared by independently pairing,via conjugation, a plurality of signal generating moieties and aplurality of second oligonucleotide sequences; iii) a third panelcomprising a plurality of universal adapters, said plurality ofuniversal adapters comprising oligonucleotide sequences havingindependently paired a plurality of oligonucleotide sequence segmentscomplementary to the plurality of first oligonucleotide sequences and aplurality of oligonucleotide sequence segments complementary to theplurality of second oligonucleotide sequences; iii) contacting a samplecomprising a plurality of targets with the first panel and the secondpanel, wherein: a) at least a first target having one or more distinctbindings sites is bound by one or more molecular probes from the firstpanel; and b) one or more detectable components from the second panelindependently hybridize with the one or more molecular probes bound tothe at least first target; iv) detecting the presence of the at leastfirst hybridized-target in said sample; v) determining a characteristicpanel of molecular probes and detectable components from said first andsecond panels that identifies the at least first target among theplurality of targets in said sample.

Example 6I

The panel development system of Example 5I, wherein the paneldevelopment system is characterized by one or more of the following: i)the plurality of first oligonucleotide sequences are identicaloligonucleotide sequences; ii) the plurality of first oligonucleotidesequence segments are identical oligonucleotide sequence segments; iii)the plurality of second oligonucleotide sequences comprises differentoligonucleotide sequences; and iv) the plurality of secondoligonucleotide sequence segments comprises oligonucleotide sequencesegments complementary to the plurality of different secondoligonucleotide sequences.

Example 7I

The panel development system of Example 5I or 6I, wherein the paneldevelopment system is characterized by one or more of the following: i)the plurality of first oligonucleotide sequences comprises differentoligonucleotide sequences; ii) the plurality of first oligonucleotidesequence segments comprises oligonucleotide sequence segmentscomplementary to the plurality of different first oligonucleotidesequences; iii) the plurality of second oligonucleotide sequences areidentical oligonucleotide sequences; and iv) the plurality of secondoligonucleotide sequence segments are identical oligonucleotide sequencesegments.

Example 8I

The panel development system of any one of Examples 5I-7I, wherein theplurality of first oligonucleotide sequences in said first panelcomprises between 2-40 different oligonucleotide sequences among theplurality of first oligonucleotide sequences.

Example 9I

The panel development system of any one of Examples 5I-8I, wherein theplurality of second oligonucleotide sequences in said second panelcomprises between 2-40 different second oligonucleotide sequences amongthe plurality of second oligonucleotide sequences.

Example 10I

The panel development system of any one of Examples 5I-9I, wherein thepanel development system further comprises a re-probe panel comprising asecond plurality of detectable components, said second plurality ofdetectable components prepared by independently pairing, viaconjugation, a second plurality of signal generating moieties and asecond plurality of second oligonucleotide sequences.

Example 11I

The panel development system of any one of Examples 1I-10I, wherein thepanel development system further comprises removing the hybridizedplurality of detectable components from the bound plurality of targets,wherein said removal is by a washing or stripping process.

Example 12I

The panel development system of any one of Examples 1I-11I, wherein thepanel development system further comprises a re-probing step comprisingthe contacting of the washed plurality of bound targets with saidre-probe panel, followed by an additional detection step anddetermination of a characteristic panel of molecular probes anddetectable components from said first, second, and re-probe panels thatidentifies the at least first target among the plurality of targets insaid sample.

Example 13I

The panel development system of any one of Examples 1I-12I, wherein theplurality of binding moieties in said first panel comprises between 2-40different binding moieties among the plurality of binding moieties.

Example 14I

The panel development system of any one of Examples 1I-13I, wherein theplurality of signal generating moieties in said second panel comprisesbetween 2-40 different signal generating moieties among the plurality ofsignal generating moieties.

Example 15I

The panel development system of any one of Examples 1I-14, wherein: i)the panel development system develops a singleplex or multiplex assay;and ii) the panel development system and the assay developed by thepanel development system detects, measures, or quantifies the level ofbinding and/or amount of the target present in the sample with one ormore of the following systems: flow cytometry, immunomagnetic cellulardepletion, immunomagnetic cell capture, array, bead array, multiplexbead array, microarray, antibody array, cellular array,chemiluminescence, infrared, microscopy, imaging, high content screening(HCS), mass cytometry, lateral flow immunoassay, immunodetection,immunohistochemistry (IHC), immunocytochemistry (ICC), in situhybridization (ISH), enzyme immuno-assay (EIA), enzyme linkedimmuno-assay (ELISA), ELISpot, immunoturbidity, latex agglutination,gold particle agglutination, visual inspection, a change in lighttransmittance through said sample, increased light transmittance throughsaid sample, a blotting method, a Western blot, a Southern blot, aSouthwestern blot, labeling inside an electrophoresis system, labelingon a surface, labeling on an array, PCR amplification, elongationfollowed by PCR amplification, immunoprecipitation,co-immunoprecipitation, chromatin immunoprecipitation, pretargetingimaging, therapeutic agent, or combinations thereof.

Example 16I

The panel development system of any one of Examples 1I-15I, wherein thepanel development system is a multiplexed panel development.

Example 17I

The panel development system of any one of Examples 1I-16I, wherein thesample comprises a plurality of cells or cell types, comprising tissuecells, cells cultured in vitro, recombinant cells, infected cells, cellsfrom laboratory animals, cells from mammal patients, cells from humanpatients, mesenchemal stem cells, stem cells, immuno-competent cells,adipose cells, fibroblasts, natural-killer cells (NK-cells), monocytes,lymphocytes, lymph node cells, T-cells, B-cells, exudate cells, effusioncells, cancer cells, blood cells, red blood cells, leukocytes, whiteblood cells, organ cells, skin cells, liver cells, splenocytes, kidneycells, intestinal cells, lung cells, heart cells, or neuronal cells.

Example 18I

The panel development system of any one of Examples 1I-17I, wherein thesample comprises a plurality of cells having 2 or more different celltypes.

Example 19I

The panel development system of any one of Examples 1I-18I, wherein thesample comprises a plurality of cells having 2-50 different cell types.

Example 20I

The panel development system of any one of Examples 1I-17I, wherein thesystem identifies a sub-population by assigning an immunophenotyperesulting from signal pattern generated.

Example 21I

The panel development system of any one of Examples 1I-20I, wherein thesystem further analyzes a sub-population of the plurality of cells.

Example 22I

The panel development system of any one of Examples 1I-21I, wherein thesample comprises a plurality of different targets or different targettypes.

Example 23I

The panel development system of any one of Examples 1I-22I, wherein thesample comprises a plurality of similar targets or similar target types.

Example 24I

The panel development system of any one of Examples 1I-23I, whereinamong a plurality of different targets in the sample at least a firsttarget having one or more distinct bindings sites is bound by one ormore molecular probes from the first panel.

Example 25I

The panel development system of any one of Examples 1I-24I, wherein thetargets or target types comprises cells and/or cell types.

Example 26I

The panel development system of any one of Examples 1I-25I, wherein theone or more distinct binding sites are distinct markers.

Example 27I

The panel development system of any one of Examples 1I-26I, wherein theone or more distinct binding sites are biomarkers.

Example 28I

The panel development system of any one of Examples 1I-27I, wherein thesystem identifies a sub-population of targets within the sample.

Example 29I

The panel development system of any one of Examples 1I-28I, wherein thesystem further comprises at least one of the following: a) measuring theamount of the at least first target in said sample; b) quantifying thelevel of the at least first target in said sample; and c) identifyingthe type of the at least first target in said sample.

Example 30I

The panel development system of any one of Examples 1I-29I, wherein oneor more of the plurality of signal generating moieties comprises one ormore of the following: a directly detectable signal generating moiety,an indirectly detectable signal generating moiety, a fluorescent dye, afluorophore, a fluorochrome, a chromophore, a biofluorescent protein, aluminescent species, a chemiluminescent compound, aelectrochemiluminescent label, a bioluminescent label, a phosphorescentspecies, a fluorophore labeled DNA dendrimer, Quantum Dot, a tandem dye,a FRET dye, a heavy atom, a spin label, a radioactive isotope, ananoparticle, a light scattering nanoparticle or microsphere, adiffracting particle, a polymer, a polymer particle, a bead, a solidsurface, a Raman particle, a metal particle, a stable isotope, a heavymetal chelate, a magnetic particle, a bead, an RFID tag, a microbarcodeparticle, an enzyme, an enzyme substrate, a molecule specificallyrecognized by another substance carrying a label or reacts with asubstance carrying a label, an antibody, an antibody fragment, anantigen, a nucleic acid, a nucleic acid analog, oligonucleotide,oligonucleotide analog, complementary oligonucleotide, complementaryoligonucleotide analog, a ligand, a protein, a peptide ligand, a proteinsubstrate, a receptor; a substrate, a secondary reporter, a hapten, orcombinations thereof.

Example 31I

The panel development system of any one of Examples 1I-30I, wherein thesecond panel comprises a plurality of detectable components, saidplurality of detectable components prepared by independently pairing,via conjugation, a plurality of scaffolds and a plurality ofcomplementary oligonucleotide sequences, wherein said plurality ofscaffolds comprises a plurality of signal generating moieties.

Example 32I

The panel development system of any one of Examples 1I-31I, wherein thescaffold comprises a dendrimer, a polysaccharide molecule, a dextran, aprotein, a peptide, a second oligonucleotide sequence, a portion of theoligonucleotide sequence that is not complementary to theoligonucleotide sequence of the molecular probe, a bead, a polymer, ahydrophilic polymer, a bead, a nanoparticle, or combinations orderivatives thereof.

Example 33I

The panel development system of any one of Examples 1I-32I, wherein thefirst panel comprises at least two different molecular probes.

Example 34I

The panel development system of any one of Examples 1I-33I, wherein thefirst panel comprises at least 2-10 different molecular probes.

Example 35I

The panel development system of any one of Examples 1I-34I, wherein thebinding moiety comprises an antibody, a monoclonal antibody, apolyclonal antibody, an enzyme, a protein, a peptide, a carbohydrate, anuclear receptor, a small molecule, an aptamer, a chelator, orcombinations or derivatives thereof.

Example 36I

The panel development system of any one of Examples 1I-35I, wherein thesecond panel comprises at least two different detectable components.

Example 37I

The panel development system of any one of Examples 1I-36I, wherein thesecond panel comprises at least 2-10 different detectable components.

Example 38I

The panel development system of any one of Examples 1I-37I, wherein thesample comprises at least two different targets.

Example 39I

The panel development system of any one of Examples 1I-38I, wherein thesample comprises at least 2-50 different targets.

Example 40I

The panel development system of any one of Examples 1I-39I, wherein thepanel development system comprises an automated system or roboticsystem.

Example 41I

A multiplexed flow cytometry assay method, comprising: i) contacting asample comprising a plurality of cells with a first series of molecularprobes and a second series of detectable components, wherein theplurality of cells comprises at least 5 different cell types; ii)binding protein markers on the plurality of cells with said firstseries; iii) hybridizing the first series or the protein marker-boundfirst series with the second series; and iv) optionally, identifying thecell types in the sample by assigning of immunophenotypes resulting fromthe hybridization of said protein marker-bound first series and saidsecond series.

Example 42I

The method of Example 41, further comprising: i) preparing said firstseries, wherein said first series comprises at least 4 differentmolecular probes, by independently pairing, via conjugation, at least 4different binding moieties and at least 4 different oligonucleotidesequences; and ii) preparing said second series, wherein said secondseries comprises at least 4 different detectable components, byindependently pairing, via conjugation, at least 4 different signalgenerating moieties and at least 4 different oligonucleotide sequencescomplementary to the sequences conjugated the binding moieties.

Example 43I

A panel development system, comprising: i) a plurality of molecularprobes comprising: a) at least a first molecular probe comprising afirst binding moiety conjugated to a first oligonucleotide sequence; andb) at least a second molecular probe comprising a second binding moietyconjugated to a second oligonucleotide sequence; ii) a plurality ofdetectable components, comprising: a) at least a first detectablecomponent comprising a first signal generating moiety conjugated to afirst complementary oligonucleotide sequence; and b) at least a seconddetectable component comprising a second signal generating moietyconjugated to a second complementary oligonucleotide sequence; and iii)providing a sample comprising a plurality of targets having at least afirst target and at least a second target; iv) binding the at least afirst target and the at least a second target of the plurality oftargets with the first binding moiety of the at least first molecularprobe and the second binding moiety of the at least second molecularprobe, respectively; v) hybridizing the at least first molecular probewith the at least first detectable component and the at second molecularprobe with the at least second detectable component, respectively; vi)detecting the at least first bound-target and the at least secondbound-target in said sample, said detection comprising at least one ofthe following: a) detecting the presence of the at least first targetand/or the at least second target in said sample; b) measuring theamount of the at least first target and/or the at least second target insaid sample; c) quantifying the level of the at least first targetand/or the at least second target in said sample; and d) identifying thetype of the at least first target and/or the at least second target insaid sample; and vii) assigning the phenotypes of the at least firsttarget and the at least second target in said sample.

Example 44I

A panel development system, comprising: i) a panel of molecular probescomprising a plurality of molecular probes, said plurality of molecularprobes comprising: a) a plurality of binding moieties comprising atleast a first binding moiety and at least a second binding moiety; b) aplurality of oligonucleotide sequences comprising at least a firstoligonucleotide sequence and at least a second oligonucleotide sequence;and c) preparing the panel of plurality of molecular probes, byindependently pairing, via conjugation, the plurality of bindingmoieties and the plurality of oligonucleotide sequences; ii) a panel ofdetectable components comprising a plurality of detectable components,comprising: a) a plurality of signal generating moieties comprising atleast a first signal generating moiety and at least a second signalgenerating moiety; b) a plurality of complementary oligonucleotidesequences comprising at least a first oligonucleotide sequencecomplementary to the at least first oligonucleotide sequence of theplurality of molecular probes and at least a second oligonucleotidesequence complementary to the at least second oligonucleotide sequenceof the plurality of molecular probes; and c) preparing the panel ofplurality of detectable components, by independently pairing, viaconjugation, the plurality of signal generating moieties and theplurality of complementary oligonucleotide sequences; iii) providing asample comprising a plurality of targets having at least a first targetand at least a second target; iv) binding the at least a first targetand the at least a second target of the plurality of targets with the atleast first binding moiety and the at least second binding moiety of thepanel of plurality of molecular probes, respectively; v) hybridizing thetarget-bound plurality of molecular probes with the panel of pluralityof detectable components; vi) detecting the presence of the at leastfirst bound-target and/or the at least second bound-target in saidsample, said detection optionally further comprising at least one of thefollowing: a) measuring the amount of the at least first target and/orthe at least second target in said sample; b) quantifying the level ofthe at least first target and/or the at least second target in saidsample; and c) identifying the type of the at least first target and/orthe at least second target in said sample; vii) assigning the phenotypeof the at least first target and the at least second target,respectively, in said sample; and viii) from the assigned phenotypes,selecting at least one of the following: a) the series of molecularprobes from the plurality of molecular probes and the series ofdetectable components from the plurality of detectable components thatdistinguish the at least first target from the at least second target insaid plurality of targets; and b) the series of molecular probes fromthe plurality of molecular probes and the series of detectablecomponents from the plurality of detectable components that distinguishthe at least second target the at least first target in said pluralityof targets.

Example 45I

A panel development system, comprising: i) a first panel comprising aplurality of molecular probes, said plurality of molecular probesprepared by independently pairing, via conjugation, a plurality ofbinding moieties and a plurality of oligonucleotide sequences; ii) asecond panel comprising a plurality of detectable components, saidplurality of detectable components prepared by independently pairing,via conjugation, a plurality of signal generating moieties and aplurality of complementary oligonucleotide sequences; iii) providing asample comprising a plurality of targets having at least a first target;iv) binding the at least a first target of the plurality of targets withat least a first binding moiety from the first panel; v) hybridizing theat least first target-bound molecular probe with the second panel; vi)detecting the presence of the at least first hybridized-target in saidsample, said detection optionally further comprising at least one of thefollowing: a) measuring the amount of the at least first target in saidsample; b) quantifying the level of the at least first target in saidsample; and c) identifying the type of the at least first target in saidsample; vii) assigning the phenotype of the at least first target insaid sample; and viii) from the assigned phenotype, selecting the seriesof molecular probes from the first panel and the series of detectablecomponents from the second panel that distinguish the at least firsttarget from the plurality of targets.

Example 46I

A panel development system, comprising: i) a first panel comprising aplurality of molecular probes, said plurality of molecular probesprepared by independently pairing, via conjugation, a plurality ofbinding moieties and a plurality of oligonucleotide sequences; ii) asecond panel comprising a plurality of detectable components, saidplurality of detectable components prepared by independently pairing,via conjugation, a plurality of signal generating moieties and aplurality of complementary oligonucleotide sequences; iii) providing asample comprising a plurality of targets having at least a first targetand at least a second target; iv) binding the at least a first targetand the at least a second target of the plurality of targets with atleast a first binding moiety and at least a second binding moiety,respectively, of the first panel; v) hybridizing the at least firsttarget-bound molecular probe and the at least second target-boundmolecular probe of the plurality of target-bound molecular probes withthe second panel; vi) detecting the presence of the at least firsthybridized-target and/or the at least second hybridized-target in saidsample, said detection optionally further comprising at least one of thefollowing: a) measuring the amount of the at least first target and/orthe at least second target in said sample; b) quantifying the level ofthe at least first target and/or the at least second target in saidsample; and c) identifying the type of the at least first target and/orthe at least second target in said sample; vii) assigning the phenotypeof the at least first target and/or the at least second target,respectively, in said sample; and viii) from the assigned phenotypes,selecting at least one of the following: a) the series of molecularprobes from the first panel and the series of detectable components fromthe second panel that distinguish the at least first target from the atleast second target in said plurality of targets; and b) the series ofmolecular probes from the first panel and the series of detectablecomponents from the second panel that distinguish the at least secondtarget the at least first target in said plurality of targets.

Example 1J

A method for assaying a target of a sample, comprising: i) providing tothe sample: 1) a molecular probe, comprising a binding moiety conjugatedto an oligonucleotide sequence; and 2) a detectable component,comprising a signal generating moiety conjugated to an oligonucleotidesequence complementary to the oligonucleotide sequence of the molecularprobe; ii) binding the target in the sample with the binding moiety ofthe molecular probe; iii) hybridizing the oligonucleotide sequence ofthe molecular probe with the complementary oligonucleotide sequence ofthe detectable component; and iv) detecting a signal generated from thehybridized detectable component; wherein the method is characterized byone or more of the following: a) the conjugation between theoligonucleotide sequence and the binding moiety and conjugation betweenthe complementary oligonucleotide sequence and the signal generatingmoiety, comprises one or more covalent bond linkages, comprising ahydrazone, oxime, triazine, or other covalent bond, wherein theformation of the conjugates are at least 90% efficient; and b) thebinding moiety comprises a binding affinity of less than 10⁻⁴ M for thetarget.

Example 2J

The method of Example 1J, wherein the mode of addition comprises: i) themolecular probe and the detectable component are combined together andhybridized prior to contacting the sample; ii) the molecular probe iscombined with the sample prior to the addition of the detectablecomponent; or iii) the detectable component is combined with the sampleprior to the addition of the molecular probe.

Example 3J

The method of any one of Examples 1I-2J, wherein the method comprises:i) the molecular probe binding the target prior to hybridizing with thedetectable component; or ii) the molecular probe hybridizing with thedetectable component prior to binding the target.

Example 4J

A method for assaying a target of a sample, comprising: i) providing tothe sample: 1) a molecular probe, comprising a binding moiety conjugatedto a first oligonucleotide sequence; 2) a detectable component,comprising a signal generating moiety conjugated to a secondoligonucleotide sequence; and 3) a universal adapter, comprising anoligonucleotide sequence having a first sequence segment complementaryto the first oligonucleotide sequence of the molecular probe and asecond sequence segment complementary to the second oligonucleotidesequence of the detectable component; ii) binding the target in thesample with the binding moiety of the molecular probe; iii) hybridizingthe first oligonucleotide sequence of the molecular probe to the firstoligonucleotide sequence segment of the universal adapter; iv)hybridizing the second oligonucleotide sequence of the detectablecomponent to the second oligonucleotide sequence segment of theuniversal adapter; and v) detecting a signal generated from thehybridized detectable component; wherein the method is characterized byone or more of the following: a) the conjugation between the firstoligonucleotide sequence and the binding moiety and conjugation betweenthe second complementary oligonucleotide sequence and the signalgenerating moiety, comprises one or more covalent bond linkages,comprising a hydrazone, oxime, triazine, or other covalent bond, whereinthe formation of the conjugates are at least 90% efficient; and b) thebinding moiety comprises a binding affinity of less than 10⁻⁴ M for thetarget.

Example 5J

The method of Example 4J, wherein the mode of addition comprises: i) themolecular probe, the universal adapter, and the detectable component arecombined together and hybridized prior to contacting the sample; ii) themolecular probe and the universal adapter are combined together andhybridized prior to contacting the sample; iii) the detectable componentand the universal adapter are combined together and hybridized prior tocontacting the sample; iv) the molecular probe, alone or in combinationwith the detectable component, is combined with the sample prior to theaddition of the universal adapter; or v) the universal adapter iscombined with the sample prior to the addition of the molecular probeand/or the detectable component.

Example 6J

The method of Examples 4J or 5J, wherein the method comprises: i) themolecular probe hybridizing with the universal adapter prior to saidmolecular probe binding the target; ii) the molecular probe hybridizingwith the universal adapter after said molecular probe binds the target;iii) the detectable component hybridizing with the universal adapterprior to the molecular probe binding the target; iv) the detectablecomponent hybridizing with the universal adapter after the molecularprobe binds the target; v) the universal adapter hybridizing with themolecular probe and hybridizing with the detectable component prior tosaid molecular probe binding the target; or vi) the universal adapterhybridizing with the molecular probe and hybridizing with the detectablecomponent after said molecular probe binds the target.

Example 7J

The method of any one of Examples 1I-6J, wherein: i) the assay comprisesa singleplex or multiplex assay; and ii) the assay detects, measures, orquantifies the level of binding and/or amount of the target present inthe sample with one or more of the following: flow cytometry,immunomagnetic cellular depletion, immunomagnetic cell capture, array,bead array, multiplex bead array, microarray, antibody array, cellulararray, chemiluminescence, infrared, microscopy, imaging, high contentscreening (HCS), mass cytometry, lateral flow immunoassay,immunodetection, immunohistochemistry (IHC), immunocytochemistry (ICC),in situ hybridization (ISH), enzyme immuno-assay (EIA), enzyme linkedimmuno-assay (ELISA), ELISpot, immunoturbidity, latex agglutination,gold particle agglutination, visual inspection, a change in lighttransmittance through said sample, increased light transmittance throughsaid sample, a blotting method, a Western blot, a Southern blot, aSouthwestern blot, labeling inside an electrophoresis system, labelingon a surface, labeling on an array, PCR amplification, elongationfollowed by PCR amplification, immunoprecipitation,co-immunoprecipitation, chromatin immunoprecipitation, pretargetingimaging, therapeutic agent, or combinations thereof.

Example 8J

The method of any one of Examples 1J-7J, wherein: i) the assay comprisesa singleplex or multiplex assay; and ii) the assay detects, measures, orquantifies the level of binding and/or amount of the target present inthe sample with one or more of the following: flow cytometry,microscopy, imaging, high content screening (HCS), multiplex bead array,microarray, antibody array, cellular array, immunohistochemistry (IHC),immunocytochemistry (ICC), in situ hybridization (ISH), enzymeimmuno-assay (EIA), enzyme linked immuno-assay (ELISA), ELISpot, or ablotting method.

Example 9J

The method of any one of Examples 1J-8J, wherein: i) the assay comprisesa singleplex or multiplex assay; and ii) the assay detects, measures, orquantifies the level of binding and/or amount of the target present inthe sample with flow cytometry.

Example 10J

The method of any one of Examples 1J-9J, wherein the sample ischaracterized as at least one or more of the following: i) a complexsample; and ii) a homogeneous or a heterogeneous mixture; and whereinsaid sample comprises at least one or more of the following: a) a rangeof analytes having a wide range of binding specificities; b) a cell, amembrane, a biological molecule, a metabolite, or a disease biomarker;and c) a biological fluid or a fluidized biological tissue.

Example 11J

The method of any one of Examples 1J-10J, wherein the hybridizationefficiency of the oligonucleotide sequence to the complementaryoligonucleotide sequence is at least 50% with respect to the detectablecomponent, under the hybridization conditions employed.

Example 12J

The method of any one of Examples 1J-11J, wherein the molecular probecomprises one or more of the following properties: i) a molecular weightof between about 15,000 Daltons to about 450,000 Daltons; ii) asolubility that is substantially the same as that of the unconjugatedbinding moiety; iii) a solubility that minimizes non-specific binding tothe target; iv) the oligonucleotide sequence of the molecular probe doesnot adversely affect the solubility of the binding moiety; v) interactsand binds to the target via interactions other than exclusivelyelectrostatic; vi) a unique, distinguishable, and/or specificallydesigned oligonucleotide sequence; and vii) the oligonucleotide sequenceof the molecular probe is uniquely and specifically designed tohybridize to the complementary oligonucleotide sequence of thedetectable component.

Example 13J

The method of any one of Examples 1J-12J, wherein the method ofdetection generates less false positives than secondary antibodydetection methods.

Example 14J

The method of any one of Examples 1I-13J, wherein the method furthercomprises: i) preparing the molecular probe; and ii) preparing thedetectable component; wherein the prepared molecular probe and prepareddetectable component have at least 90% purity.

Example 15J

The method of any one of Examples 1J-14J, wherein the method furthercomprises preparing and isolating the molecular probe, comprising: i)providing the binding moiety; ii) conjugating the binding moiety with atleast one oligonucleotide at greater than 90% efficiency to form bindingmoiety-oligonucleotide conjugates; and iii) isolating the bindingmoiety-oligonucleotide conjugates from the conjugation mixture bybinding, retaining, and/or retarding a substantial portion of: a) theconjugates, removing a substantial portion of the unconjugatedoligonucleotide in a wash step followed by release of the bound,retained, and/or retarded conjugates; or b) the unconjugatedoligonucleotides, followed by collecting a substantial portion of thenon-bound, non-retained, and/or non-retarded conjugates in a wash step.

Example 16J

The method of any one of Examples 1J-15J, wherein the method furthercomprises preparing and isolating the detectable component, comprising:i) providing a plurality of the signal generating moiety; ii)conjugating the complementary oligonucleotide with at least one of theplurality of the signal generating moiety at greater than 90% efficiencyto form signal generating moiety-complementary oligonucleotideconjugates; and iii) isolating the signal generatingmoiety-complementary oligonucleotide conjugates from the conjugationmixture by binding, retaining, and/or retarding a substantial portionof: a) the conjugates, removing a substantial portion of theunconjugated oligonucleotide in a wash step followed by release of thebound, retained, and/or retarded conjugates; or b) the unconjugatedoligonucleotides, followed by collecting a substantial portion of thenon-bound, non-retained, and/or non-retarded conjugates in a wash step.

Example 17J

The method of any one of Examples 1J-16J, wherein the detectablecomponent comprises a scaffold conjugated to the complementaryoligonucleotide sequence, and wherein said scaffold has one or moresignal generating moieties.

Example 18J

The method of Example 17J, wherein the scaffold comprises a dendrimer, apolysaccharide, a dextran, a protein, a peptide, a secondoligonucleotide sequence, a portion of the oligonucleotide sequence thatis not complementary to the oligonucleotide sequence of the molecularprobe, a polymer, a hydrophilic polymer, a bead, a nanoparticle, orcombinations or derivatives thereof.

Example 19J

The method of any one of Examples 1J-18J, wherein the method furthercomprises preparing and isolating a detectable component comprising ascaffold conjugated to the oligonucleotide sequence complementary to theoligonucleotide sequence of the molecular probe, wherein the scaffoldcomprises one or more signal generating moieties, said methodcomprising: i) providing a plurality of the scaffold comprising the oneor more signal generating moieties; ii) conjugating the complementaryoligonucleotide with at least one of the plurality of scaffolds atgreater than 90% efficiency to form scaffold-complementaryoligonucleotide conjugates; and iii) isolating thescaffold-complementary oligonucleotide conjugates from the conjugationmixture by binding, retaining, and/or retarding a substantial portionof: a) the conjugates, removing a substantial portion of theunconjugated oligonucleotide in a wash step followed by release of thebound, retained, and/or retarded conjugates; or b) the unconjugatedoligonucleotides, followed by collecting a substantial portion of thenon-bound, non-retained, and/or non-retarded conjugates in a wash step.

Example 20J

The method of any one of Examples 15J-19J, wherein the isolation steputilizes an immobilized binder, affinity chromatography, size exclusionchromatography, HPLC, reverse-phase chromatography, electrophoresis,capillary electrophoresis, polyacrylamide gel electrophoresis, agarosegel electrophoresis, free flow electrophoresis, differentialcentrifugation, thin layer chromatography, immunoprecipitation,hybridization, solvent extraction, dialysis, filtration, diafiltration,tangential flow filtration, ion exchange chromatography, hydrophobicinteraction chromatography, or combinations thereof.

Example 21J

The method of any one of Examples 15J-19J, wherein the isolation steputilizes an immobilized binder, chromatography, or size exclusionchromatography.

Example 22J

The method of any one of Examples 1J-21J, wherein the binding moietycomprises an antibody, a monoclonal antibody, a polyclonal antibody, anenzyme, a protein, a peptide, a carbohydrate, a nuclear receptor, asmall molecule, an aptamer, a chelator, or combinations or derivativesthereof.

Example 23J

The method of any one of Examples 1J-22J, wherein the sample comprisesone or more targets.

Example 24J

The method of any one of Examples 1J-23J, wherein the target is abiological target.

Example 25J

The method of Example 24J, wherein the biological target comprises anantigen, a pathogen, a protein, a peptide, an epitope, acarbohydrate-containing molecule, a small molecule, or combinations orderivatives thereof.

Example 26J

The method of any one of Examples 1J-25J, wherein the one or more signalgenerating moieties, comprise one or more of the following: a directlydetectable signal generating moiety, an indirectly detectable signalgenerating moiety, a fluorescent dye, a fluorophore, a fluorochrome, achromophore, a fluorescent protein, a biofluorescent protein, aluminescent species, a chemiluminescent compound, aelectrochemiluminescent label, a bioluminescent label, a phosphorescentspecies, a fluorophore labeled DNA dendrimer, Quantum Dot, a Ramanparticle, a tandem dye, a FRET dye, a heavy atom, a spin label, aradioactive isotope, a nanoparticle, a light scattering nanoparticle ormicrosphere, a diffracting particle, a polymer, a polymer particle, abead, a solid surface, a metal particle, a stable isotope, a heavy metalchelate, a magnetic particle, an RFID tag, a microbarcode particle, anenzyme, an enzyme substrate, a molecule specifically recognized byanother substance carrying a label or reacts with a substance carrying alabel, an antibody, an antibody fragment, an antigen, a nucleic acid, anucleic acid analog, oligonucleotide, oligonucleotide analog,complementary oligonucleotide, complementary oligonucleotide analog, aligand, a protein, a peptide ligand, a protein substrate, a receptor; asubstrate, a secondary reporter, a hapten, or combinations orderivatives thereof.

Example 27J

The method of any one of Examples 1J-26J, wherein the one or more signalgenerating moieties provides an enhanced signal that minimizes detectionerrors from background noise, relative to conventionally labeled bindingmoieties.

Example 28J

The method of any one of Examples 1J-27J, wherein the molecular probe,the detectable component, and/or universal adapter further comprises aspacer group, comprising a polymerized ethylene oxide, a PEG, a PEO, aprotein, a peptide, a DNA, an RNA, an oligonucleotide sequence, or adextran.

Example 29J

The method of any one of Examples 1J-28J, wherein the binding moiety,the scaffold, the oligonucleotide sequence, and/or the complementaryoligonucleotide sequence comprise HyNic or 4-FB.

Example 30J

The method of any one of Examples 1J-29J, wherein the detectablecomponent comprises a unique, distinguishable, and/or specificallydesigned complementary oligonucleotide sequence.

Example 31J

The method of any one of Examples 1J-30J, wherein the oligonucleotidesequence, complementary oligonucleotide sequence, and/or oligonucleotidesequence segment comprises an oligonucleotide sequence conjugated at the3′-position, an oligonucleotide sequence conjugated at the 5′-position,linear oligonucleotide sequences, branched oligonucleotide sequences,LNAs, PNAs, oligonucleotide sequences optionally covalently attached toother moieties, or combinations or derivatives thereof.

Example 32J

The method of any one of Examples 1J-31J, wherein the sample comprisesone or more targets, and to sample is provided: i) a plurality ofmolecular probes, comprising at least a first molecular probe and atleast a second molecular probe; and ii) a plurality of detectablecomponents, comprising at least a first detectable component and atleast a second detectable component.

Example 33J

The method of any one of Examples 1J-32J, wherein the method furthercomprises binding the one or more targets, comprising at least one ofthe following: i) binding at least a first target of the one or moretargets in the sample with the first binding moiety of the at leastfirst molecular probe; and ii) binding at least a second target of theone or more targets in the sample with the second binding moiety of theat least second molecular probe.

Example 34J

The method of any one of Examples 1J-33J, wherein the method furthercomprises hybridizing the plurality of molecular probes and theplurality of detectable components, comprising at least one of thefollowing: i) hybridizing the first oligonucleotide sequence of at leastfirst molecular probe to the first complementary oligonucleotidesequence segment of the at least first detectable component; and ii)hybridizing the second oligonucleotide sequence of at least secondmolecular probe to the second complementary oligonucleotide sequencesegment of the at least second detectable component.

Example 35J

The method of any one of Examples 1J-34J, wherein the method furthercomprises detecting one or more signals generated from at least one ofthe following: i) the at least first hybridized detectable component;and ii) the at least second hybridized detectable component.

Example 36J

The method of any one of Examples 1J-35J, wherein the conjugationbetween the first oligonucleotide sequence and the first binding moiety,between the second oligonucleotide sequence and the second bindingmoiety, between the first complementary oligonucleotide sequence and thefirst signal generating moiety, and between the second complementaryoligonucleotide sequence and the second signal generating moiety,comprises one or more covalent bond linkages, comprising a hydrazone,oxime, triazine, or other covalent bond, wherein the formation of theconjugates are at least 90% efficient.

Example 37J

The method of any one of Examples 1J-36J, wherein the first bindingmoiety comprises a binding affinity of less than 10⁻⁴ M for the at leastfirst target of the one or more targets and the second binding moietycomprises a binding affinity of less than 10⁻⁴ M for the at least secondtarget of the one or more targets.

Example 38J

The method of any one of Examples 1I-37J, wherein the detectablecomponent comprises a bead conjugated to the second oligonucleotidesequence or the complementary second oligonucleotide sequence, andwherein said bead comprises one or more signal generating moieties.

Example 39J

The method of any one of Examples 1J-38J, wherein: i) the at least firstdetectable component comprises a first bead; and/or ii) the at leastsecond detectable component comprises a second bead; wherein said firstbead and second bead comprise one or more signal generating moieties.

Example 40J

A method for crosslinking, comprising: i) introducing to a samplecomprising one or more targets: a) one or more firstantibody-oligonucleotide conjugates, comprising a first antibodyconjugated to a first oligonucleotide sequence; and b) one or moresecond antibody-oligonucleotide conjugates, comprising a second antibodyconjugated to a second oligonucleotide sequence; ii) binding the one ormore targets with the first antibody of the one or more firstantibody-oligonucleotide conjugates and with the second antibody of theone or more second antibody-oligonucleotide conjugates to form one ormore sandwich-complexes; iii) contacting the one or moresandwich-complexes with: a) one or more first bead-oligonucleotideconjugate, comprising a first bead conjugated to a complementary firstoligonucleotide sequence; and b) one or more second bead-oligonucleotideconjugate, comprising a second bead conjugated to a complementary secondoligonucleotide sequence; iv) crosslinking the one or moresandwich-complexes by: a) hybridizing the first oligonucleotidesequences of the one or more sandwich-complexes with the complementaryfirst oligonucleotide sequences of the one or more firstbead-oligonucleotide conjugates; and b) hybridizing the secondoligonucleotide sequences of the one or more sandwich-complexes with thecomplementary second oligonucleotide sequences of the one or more secondbead-oligonucleotide conjugates.

Example 41J

The crosslinking method of Example 40J, wherein the formation of thecrosslinked one or more sandwich-complexes forms an agglutination.

Example 42J

The crosslinking method of Examples 40J or 41J, wherein the methodfurther comprises detecting, measuring, and/or quantifying the degree ofthe formed agglutination to determine the amount of the one or moretargets in the sample.

Example 43J

The crosslinking method of any one of Examples 40J-42J, wherein thefirst antibody or the second antibody comprise a monoclonal antibody ora polyclonal antibody.

Example 44J

The crosslinking method of any one of Examples 40J-43J, wherein: i) thefirst antibody comprises a first polyclonal antibody and the secondantibody comprises a second polyclonal antibody; ii) the first antibodycomprises a first monoclonal antibody and the second antibody comprisesa second monoclonal antibody; iii) the first antibody comprises a firstmonoclonal antibody and the second antibody comprises a first polyclonalantibody; or iv) the first antibody comprises a first polyclonalantibody and the second antibody comprises a first monoclonal antibody.

Example 45J

The crosslinking method of any one of Examples 40J-44J, wherein thefirst antibody comprises a first monoclonal antibody and the secondantibody comprises a second monoclonal antibody.

Example 46J

The crosslinking method of any one of Examples 40J-44J, wherein thefirst antibody comprises a first polyclonal antibody and the secondantibody comprises a second polyclonal antibody.

Example 47J

The crosslinking method of any one of Examples 40J-44J, wherein thefirst antibody comprises a first monoclonal antibody and the secondantibody comprises a first polyclonal antibody.

Example 48J

The crosslinking method of any one of Examples 40J-47J, wherein thedetection comprises a singleplex or multiplex detection, comprising:immunodetection, immunoturbidity, latex agglutination, gold particleagglutination, visual inspection, a change in light transmittancethrough said sample, increased light transmittance through said sample,flow cytometry, microscopy, imaging, high content screening (HCS),immunohistochemistry, ELISA, ELISpot, arrays, bead arrays, orcombinations or derivatives thereof.

Example 49J

A method of preparing a detectable component having one or moresignal-generating moieties, comprising: i) modifying a scaffold withS-HyNic to form a HyNic-modified scaffold; ii) conjugating a4FB-modified oligonucleotide to the HyNic-modified scaffold, wherein theconjugation is at least 90% efficient; and iii) modifying the scaffoldof the oligonucleotide-scaffold conjugate with one or moresignal-generating moieties to form the detectable component.

Example 50J

A method of preparing one or more detectable components, comprising: i)modifying one or more scaffolds; ii) conjugating one or more modifiedoligonucleotides to the one or more modified scaffolds, wherein theconjugation is at least 90% efficient; and iii) modifying the scaffoldof the one or more oligonucleotide-scaffold conjugates with one or moresignal-generating moieties to form one or more components; wherein theconjugation between the one or more modified-oligonucleotides and theone or more modified scaffolds comprises one or more covalent bondlinkages, comprising a hydrazone, oxime, triazine, or other covalentbond.

Example 51J

The preparation method of Examples 49J or 50J, wherein the one or morescaffolds comprises: a hydrophilic polymer, a dendrimer, apolysaccharide, a dextran, a protein, a peptide, a secondoligonucleotide sequence, a portion of the oligonucleotide sequence thatis not complementary to the oligonucleotide sequence of the molecularprobe, a bead, a nanoparticle, or combinations thereof.

Example 52J

The preparation method of any one of Examples 49J-51J, wherein thehydrophilic polymer comprises a polysaccharide molecule.

Example 53J

The preparation method of Example 52J, wherein the polysaccharidemolecule comprises a dextran or an amino-dextran.

Example 54J

The preparation method of any one of Examples 49J-53J, wherein the oneor more signal-generating moieties comprises a directly detectablesignal-generating moiety or an indirectly detectable signal-generatingmoiety.

Example 55J

The preparation method of Example 54J, wherein the directly detectablesignal-generating moiety comprises: a fluorescent dye; a luminescentspecies; a phosphorescent species; a radioactive substance; ananoparticle; a diffracting particle; a raman particle; a metalparticle; a magnetic particle; a bead; an RFID tag; a microbarcodeparticle; or combinations thereof.

Example 56J

The preparation method of any one of Examples 49J-55J, wherein theindirectly detectable signal-generating moiety comprises: an enzyme; anantibody; an antigen; a nucleic acid; a nucleic acid analog;oligonucleotide; oligonucleotide analog; complementary oligonucleotide;complementary oligonucleotide analog; a ligand; a substrate; a hapten;or combinations thereof.

Example 57J

The preparation method of any one of Examples 49J-56J, wherein the oneor more scaffolds signal-generating moieties comprise: a fluorophore; achromophore; a biofluorescent protein; a fluorophore labeled DNAdendrimer; a Quantum Dot; a chemiluminescent compound; aelectrochemiluminescent label; a bioluminescent label; a polymer; apolymer particle; a bead; a Raman particle; a heavy metal chelate; goldor other metal particles or heavy atoms; a spin label; a radioactiveisotope; a secondary reporter; a hapten; a nucleic acid or nucleic acidanalog; oligonucleotide; oligonucleotide analog; complementaryoligonucleotide; complementary oligonucleotide analog; a protein; apeptide ligand or substrate; a receptor; an enzyme; an enzyme substrate;an antibody; an antibody fragment; an antigen; or combinations orderivatives thereof.

Example 58J

The method of any one of Examples 1J-57J, wherein the plurality oftargets comprises a plurality of cells, said plurality of cellscomprising at least a first cell and at least a second cell.

Example 59J

The method of any one of Examples 1J-58J, wherein at least a firsttarget comprises a first biomarker of the at least first cell and atleast a second target comprises a second biomarker of the at leastsecond cell.

Example 60J

The method of any one of Examples 1J-59J, wherein the first biomarkercomprises a protein biomarker and the second biomarker comprises aprotein biomarker.

Example 61J

The method of any one of Examples 1J-60J, wherein the first biomarkercomprises an adhesion molecule and the second biomarker comprises anadhesion molecule.

Example 62J

The method of any one of Examples 1J-61J, wherein the plurality of cellscomprises immuno-competent cells.

Example 63J

The method of any one of Examples 1J-62J, wherein the plurality of cellscomprises at least one of the following: tissue cells, cells cultured invitro, recombinant cells, infected cells, cells from laboratory animals,cells from mammal patients, cells from human patients, mesenchemal stemcells, stem cells, immuno-competent cells, adipose cells, fibroblasts,natural-killer cells (NK-cells), monocytes, lymphocytes, lymph nodecells, T-cells, B-cells, exudate cells, effusion cells, cancer cells,blood cells, red blood cells, leukocytes, white blood cells, organcells, skin cells, liver cells, splenocytes, kidney cells, intestinalcells, lung cells, heart cells, or neuronal cells.

Example 64J

The method of any one of Examples 1J-63J, wherein the first cell is aT-cell and the second cell is a B-cell.

Example 65J

A method for binding, comprising: i) incubating a plurality of cells ormaterial derived from the plurality of cells with: a) at least onemolecular probe, comprising a binding moiety conjugated to anoligonucleotide sequence; and b) at least one detectable component,comprising one or more signal generating moieties conjugated to acomplementary oligonucleotide sequence; iii) binding at least one targetfrom the plurality of cells with the binding moiety of the at least onemolecular probe; and iv) hybridizing the oligonucleotide sequence of theat least one bound molecular probe to the complementary oligonucleotidesequence of the at least one detectable component; wherein the method ischaracterized by one or more of the following: a) the conjugation of theat least one molecular probe and the conjugation of the at least onedetectable component comprises one or more covalent bond linkages,comprising a hydrazone, oxime, triazine, or other covalent bond; b) theformation of the conjugates are at least 90% efficient; and c) thebinding moiety of the at least one molecular probe has a bindingaffinity for the at least one target of less than 10⁻⁴ M.

Example 66J

The method of Examples 65J, wherein the method further comprises theaddition of at least one universal adapter comprising a firstoligonucleotide sequence segment complementary to the firstoligonucleotide sequence of the at least first molecular probe and asecond oligonucleotide sequence segment complementary to the secondoligonucleotide sequence of the at least first detectable component;wherein the first oligonucleotide sequence of the at least firstmolecular probe and the second oligonucleotide sequence of the at leastfirst detectable component are non-complementary.

Example 67J

The method of Examples 65J or 66J, wherein the at least one target fromthe plurality of cells is derived by lysing the plurality of cells.

Example 68J

The method of any one of Examples 65J-67J, wherein the at least onetarget from the plurality of cells or from the material derived from theplurality of cells is derived by at least one of the following:electrophoresing material derived from lysing the plurality of cells,secretion by the plurality of cells, biological fluids, extracellularmatrix proteins, cell culture media, genetically engineered proteins ornucleic acids produced by the plurality of cells, or food stuffs.

Example 69J

The method of any one of Examples 65J-68J, wherein the at least onemolecular probe and the at least one detectable component are hybridizedprior to incubating.

Example 70J

The method of any one of Examples 65J-69J, wherein the at least onemolecular probe and the at least one detectable component are hybridizedprior to incubating with the electrophoresed material.

Example 71J

The method of any one of Examples 65J-70J, wherein the method furthercomprises transferring the electrophoresed material to a membrane.

Example 72J

The method of any one of Examples 65J-71J, wherein the electrophoresingof the lysate comprises a Western Blot.

Example 73J

The method of any one of Examples 65J-72J, wherein the membranecomprises a PVDF membrane, a nitrocellulose membrane, or a nylonmembrane.

Example 74J

The method of any one of Examples 1J-73J, wherein the one or more signalgenerating moieties of the at least one detectable component compriseshorseradish peroxidase.

Example 75J

The method of any one of Examples 1J-74J, wherein the method comprisesdetecting the at least one signal generated from the one or more signalgenerating moieties on the at least one hybridized detectable component.

Example 76J

The method of any one of Examples 1J-75J, wherein the detectioncomprises a singleplex or multiplex detection, comprising:immunodetection, flow cytometry, chemiluminescence detection,colormetric detection, fluorescence detection, light-scatteringdetection, line-scanning, infrared detection, microscopy, imaging, highcontent screening (HCS), immunohistochemistry, ELISA, ELISpot, arrays,bead arrays, immunoturbidity, latex agglutination, gold particleagglutination, visual inspection, a change in light transmittancethrough said sample, increased light transmittance through said sample,or combinations or derivatives thereof.

Example 77J

The method of any one of Examples 1J-76J, wherein the target or the oneor more targets comprises cells, cellular components, biomarkers,biological components, or combinations thereof.

Example 78J

The method of any one of Examples 1J-77J, wherein the cells are attachedto a bead or a plate.

Example 79J

The method of any one of Examples 1J-78J, wherein the cellularcomponents comprises tubulin.

Example 80J

A method of bead crosslinking or agglutination, comprising: i)hybridizing a plurality of antibody-oligonucleotide conjugates with aplurality of bead-complementary oligonucleotide conjugates to form aplurality of hybridized antibody-bead conjugates; ii) introducing theplurality of hybridized antibody-bead conjugates to a sample comprisingone or more targets; iii) binding at least one target of the one or moretargets with at least one hybridized antibody-bead conjugate of theplurality of hybridized antibody-bead conjugates; iv) forming acrosslinking or agglutination of the at least one target-boundhybridized antibody-bead conjugate; and v) analyzing the agglutinationto detect, measure, and/or quantify the presence or amount of the atleast one target by at least one of the following: a) visual inspection;and b) decreased absorption.

Example 81J

The method of Example 80J, wherein the sample is a biological sample.

Example 82J

The method of Example 81J, wherein the least one target of the pluralityof targets comprises a biological target.

Example 83J

The method of Example 82J, wherein the biological target comprises anantigen, a pathogen, a protein, a peptide, an epitope, acarbohydrate-containing molecule, a small molecule, or combinations orderivatives thereof.

The foregoing description of certain exemplary embodiments has beenpresented for purposes of illustration and description. It is notintended to be exhaustive of, or to limit, the disclosure to the preciseform disclosed, and modification and variations are possible in light ofthe teachings herein or may be acquired from practice of the disclosedembodiments. The embodiments shown and described in order to explain theprinciples of the inventions and its practical application to enable oneskilled in the art to utilize various embodiments and with variousmodifications as are suited to the particular application contemplated.Accordingly, such modifications and embodiments are intended to beincluded within the scope of the disclosure. Other substitutions,modifications, changes and omissions may be made in the design,operating conditions, and arrangement of the exemplary embodimentwithout departing from the spirit of the present disclosure.

What is claimed is:
 1. A tunable detection system, comprising: i) amolecular probe prepared by conjugating a first oligonucleotide sequenceto a binding moiety; and ii) a series of detectable components,comprising a range of signal generating moieties conjugated to a secondoligonucleotide sequence, wherein the range of signal generatingmoieties generates a range of signal intensities, and wherein the secondoligonucleotide sequence is complementary to the first oligonucleotidesequence; wherein the range of signal intensities generated can be tunedover a range from the limit of self-quenching to the intensity of asingle signal generating moiety.
 2. A tunable detection system,comprising: i) a molecular probe prepared by conjugating a firstoligonucleotide sequence to a binding moiety; and ii) a series ofdetectable components, comprising a range of signal generating moietiesconjugated to a second oligonucleotide sequence, wherein the range ofsignal generating moieties generates a range of signal intensities; andiii) a universal adapter, comprising a first oligonucleotide sequencesegment complementary to the first oligonucleotide sequence and a secondoligonucleotide sequence segment complementary to the secondoligonucleotide sequence; wherein the range of signal intensitiesgenerated can be tuned over a range from the limit of self-quenching tointensity of a single signal generating moiety.
 3. The tunable detectionsystem of claim 1, wherein the detectable component or the hybridizeddetectable component comprises one or more signal generating moieties,comprising one or more of the following: a directly detectable signalgenerating moiety, an indirectly detectable signal generating moiety, afluorescent dye, a fluorophore, a fluorochrome, a chromophore, abiofluorescent protein, a luminescent species, a chemiluminescentcompound, a electrochemiluminescent label, a bioluminescent label, aphosphorescent species, a fluorophore labeled DNA dendrimer, QuantumDot, a tandem dye, a FRET dye, a heavy atom, a spin label, a radioactiveisotope, a nanoparticle, a light scattering nanoparticle or microsphere,a diffracting particle, a polymer, a polymer particle, a bead, a solidsurface, a Raman particle, a metal particle, a stable isotope, a heavymetal chelate, a magnetic particle, an RFID tag, a microbarcodeparticle, an enzyme, an enzyme substrate, a molecule specificallyrecognized by another substance carrying a label or reacts with asubstance carrying a label, an antibody, an antibody fragment, anantigen, a nucleic acid, a nucleic acid analog, oligonucleotide,oligonucleotide analog, complementary oligonucleotide, complementaryoligonucleotide analog, a ligand, a protein, a peptide ligand, a proteinsubstrate, a receptor; a substrate, a secondary reporter, a hapten, orcombinations thereof.
 4. The tunable detection system of claim 1,wherein the detectable component comprises a scaffold conjugated to thesecond oligonucleotide sequence, and wherein said scaffold comprises theone or more signal generating moieties.
 5. The tunable detection systemof claim 1, wherein: i) the tunable detection system comprises asingleplex or multiplex tunable detection system; and ii) the tunabledetection system detects, measures, or quantifies the level of bindingand/or amount of one or more targets present in a sample from thegenerated signal by one or more of the following: flow cytometry,immunomagnetic cellular depletion, immunomagnetic cell capture, array,bead array, multiplex bead array, microarray, antibody array, cellulararray, chemiluminescence, infrared, microscopy, imaging, high contentscreening (HCS), mass cytometry, lateral flow immunoassay,immunodetection, immunohistochemistry (IHC), immunocytochemistry (ICC),in situ hybridization (ISH), enzyme immuno-assay (EIA), enzyme linkedimmuno-assay (ELISA), ELISpot, immunoturbidity, latex agglutination,gold particle agglutination, visual inspection, a change in lighttransmittance through said sample, increased light transmittance throughsaid sample, a blotting method, a Western blot, a Southern blot, aSouthwestern blot, labeling inside an electrophoresis system, labelingon a surface, labeling on an array, PCR amplification, elongationfollowed by PCR amplification, immunoprecipitation,co-immunoprecipitation, chromatin immunoprecipitation, pretargetingimaging, therapeutic agent, or combinations thereof.
 6. A tunabledetection system, comprising: i) a plurality of molecular probescomprising: 1) at least a first molecular probe prepared by conjugatinga first oligonucleotide sequence to a first binding moiety; and 2) atleast a second molecular probe prepared by conjugating a secondoligonucleotide sequence to a second binding moiety; and ii) a pluralityof detectable components comprising: 1) at least a first detectablecomponent comprising a range of first signal generating moietiesconjugated to a oligonucleotide sequence complementary to said firstoligonucleotide sequence, wherein the range of first signal generatingmoieties generates a range of signal intensities; and 2) at least asecond detectable component comprising a range of second signalgenerating moieties conjugated to a oligonucleotide sequencecomplementary to said second oligonucleotide sequence, wherein the rangeof second signal generating moieties generates a range of signalintensities; wherein the range of signal intensities generated from theat least first detectable component and the at least second detectablecomponent can be individually tuned over a range from the limit ofself-quenching to intensity of the single first signal generating moietyor the second signal generating moiety, respectively.
 7. The tunabledetection system of claim 6, wherein: i) the first signal generated isfrom at least a first target in a sample bound by the at least firstmolecular probe that is hybridized to the at least first detectablecomponent; and ii) the second signal generated is from at least a secondtarget in the sample bound by the at least second molecular probe thatis hybridized to the at least second detectable component.
 8. Thetunable detection system of claim 6, wherein the plurality of detectablecomponents, the at least first hybridized detectable component, and/orthe at least second hybridized detectable component comprise one or moresignal generating moieties, wherein said one or more signal generatingmoieties comprises one or more of the following: a directly detectablesignal generating moiety, an indirectly detectable signal generatingmoiety, a fluorescent dye, a fluorophore, a fluorochrome, a chromophore,a biofluorescent protein, a luminescent species, a chemiluminescentcompound, a electrochemiluminescent label, a bioluminescent label, aphosphorescent species, a fluorophore labeled DNA dendrimer, QuantumDot, a tandem dye, a FRET dye, a heavy atom, a spin label, a radioactiveisotope, a nanoparticle, a light scattering nanoparticle or microsphere,a diffracting particle, a polymer, a polymer particle, a bead, a solidsurface, a Raman particle, a metal particle, a stable isotope, a heavymetal chelate, a magnetic particle, an RFID tag, a microbarcodeparticle, an enzyme, an enzyme substrate, a molecule specificallyrecognized by another substance carrying a label or reacts with asubstance carrying a label, an antibody, an antibody fragment, anantigen, a nucleic acid, a nucleic acid analog, oligonucleotide,oligonucleotide analog, complementary oligonucleotide, complementaryoligonucleotide analog, a ligand, a protein, a peptide ligand, a proteinsubstrate, a receptor; a substrate, a secondary reporter, a hapten, orcombinations thereof.
 9. The tunable detection system of claim 6,wherein the at least first hybridized detectable component comprises afirst scaffold conjugated to the first oligonucleotide sequence and/orthe at least second hybridized detectable component comprises a secondscaffold conjugated to the second oligonucleotide sequence.
 10. Thetunable detection system of claim 6, wherein the first scaffold and/orthe second scaffold comprises a dendrimer, a polysaccharide molecule, adextran, a protein, a peptide, an additional oligonucleotide sequence, aportion of the first or second oligonucleotide sequence that is notcomplementary to the first or second oligonucleotide sequence of the atleast first or second molecular probe, a polymer, a hydrophilic polymer,a bead, a nanoparticle, or combinations or derivatives thereof.
 11. Thetunable detection system of claim 6, wherein the first scaffold and/orthe second scaffold has one or more signal generating moieties.
 12. Thetunable detection system of claim 6, wherein: i) the tunable detectionsystem comprises a singleplex or multiplex tunable detection system; andii) the tunable detection system detects, measures, or quantifies thelevel of binding and/or amount of one or more targets present in asample from the signal generated from the at least first detectablecomponent and/or the signal generated from the at least seconddetectable component by one or more of the following: flow cytometry,immunomagnetic cellular depletion, immunomagnetic cell capture, array,bead array, multiplex bead array, microarray, antibody array, cellulararray, chemiluminescence, infrared, microscopy, imaging, high contentscreening (HCS), mass cytometry, lateral flow immunoassay,immunodetection, immunohistochemistry (IHC), immunocytochemistry (ICC),in situ hybridization (ISH), enzyme immuno-assay (EIA), enzyme linkedimmuno-assay (ELISA), ELISpot, immunoturbidity, latex agglutination,gold particle agglutination, visual inspection, a change in lighttransmittance through said sample, increased light transmittance throughsaid sample, a blotting method, a Western blot, a Southern blot, aSouthwestern blot, labeling inside an electrophoresis system, labelingon a surface, labeling on an array, PCR amplification, elongationfollowed by PCR amplification, immunoprecipitation,co-immunoprecipitation, chromatin immunoprecipitation, pretargetingimaging, therapeutic agent, or combinations thereof.
 13. The tunabledetection system of claim 6, wherein the tunable detection systemminimizes signal spillover by varying one or more of the following: theidentity of the first signal generating moiety, the number of the firstsignal generating moieties on the at least first detectable component,the identity of the second signal generating moiety, the number of thesecond signal generating moieties on the at least second detectablecomponent.
 14. The tunable detection system of claim 1, wherein thesample comprises a plurality of targets.
 15. The tunable detectionsystem of claim 1, wherein the tunable detection system comprises one ormore of the following: i) the plurality of molecular probes comprisesthe plurality of binding moieties independently conjugated to aplurality of oligonucleotide sequences, comprising: 1) at least a firstmolecular probe comprising the first binding moiety conjugated to afirst oligonucleotide sequence; and 2) at least a second molecular probecomprising the second binding moiety conjugated to a secondoligonucleotide sequence; and ii) the plurality of detectable componentscomprising the range of first signal generating moieties independentlyconjugated to the universal oligonucleotide sequence, comprising: 1) atleast a first detectable component comprising the range of first signalgenerating moieties conjugated to the universal oligonucleotidesequence, wherein the range of first signal generating moietiesgenerates a range of signal intensities; and 2) at least a seconddetectable component comprising the range of second signal generatingmoieties conjugated to the universal oligonucleotide sequence, whereinthe range of second signal generating moieties generates a range ofsignal intensities; and iii) a plurality of universal adapters,comprising a plurality of oligonucleotide sequence segmentscomplementary to the plurality of oligonucleotide sequence of theplurality of molecular probes independently paired with a secondoligonucleotide sequence segment complementary to the universaloligonucleotide sequence.
 16. The tunable detection system of claim 1,wherein the tunable detection system comprises an automated system orrobotic system.
 17. The tunable detection system of claim 1, wherein thetunable detection system further comprises removing the hybridizeddetectable component or plurality of detectable components from thebound target or plurality of targets, respectively, wherein said removalis by a washing or stripping process.
 18. The tunable detection systemof claim 17, wherein the tunable detection system further comprisesre-probing with a second detectable component or second plurality ofdetectable components, respectively, wherein said second detectablecomponent comprises at least one second signal generating moietiesconjugated to a second oligonucleotide sequence or a complementarysecond oligonucleotide sequence, or said second plurality of detectablecomponents are prepared by independently pairing, via conjugation, asecond plurality of signal generating moieties and a second plurality ofsecond oligonucleotide sequences or a second plurality of complementarysecond oligonucleotide sequences.
 19. The tunable detection system ofclaim 1, wherein the intensity of the signal generated can be tuned overa range 2 to 10×.
 20. The tunable detection system of claim 1, whereinthe binding moiety comprises an antibody, a monoclonal antibody, apolyclonal antibody, an enzyme, a protein, a peptide, a carbohydrate, anuclear receptor, a small molecule, an aptamer, a chelator, orcombinations or derivatives thereof.